Method of grafting genetically modified cells to treat defects, disease or damage of the central nervous system

ABSTRACT

Methods of genetically modifying donor cells by gene transfer for grafting into the central nervous system to treat defective, diseased or damaged cells are disclosed. The modified donor cells produce functional molecules that effect the recovery or improved function of cells in the CNS. Methods and vectors for carrying out gene transfer and grafting are described.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant Contract No.N00014-86-K-0347 awarded by the Office of Naval Research, and GrantContract Nos. HD-20034, NIA-06088, HD-00669, awarded by NIH and NIA R01AG08514, awarded by the Department of Health and Human Services. TheGovernment has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 792,894, filed Nov.15, 1991, now abandoned which was is a continuation-in-part of patentapplication U.S. Ser. No. 285,196, filed Dec. 15, 1988, now U.S. Pat.No. 5,082,670, the entire disclosure of which is expressly incorporatedby reference herein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the use of recombinant technology forgenetic modification of donor cells for grafting into the centralnervous system (CNS) of a subject to treat defects, disease or damage ofthe CNS. More specifically, the invention relates to the insertion ofgenes into donor cells, encoding molecules having ameliorative effectson cells in the CNS, including neurons into donor cells such that whenthe donor cells are grafted into the CNS the molecules are expressed andexert their effects on diseased or damaged cells.

BACKGROUND OF THE INVENTION

Attempts to repair the mammalian brain or replace CNS functionsresulting from defects or following disease or damage to the CNS arehampered by an incomplete understanding of the complexstructure-function relationships in the CNS. Although knowledge of somebasic principles of cell function in the brain has advanced greatly inrecent years, understanding of interactions between clusters of cells orsystems and cell circuits in different regions of the brain and theirrelationship to the outward manifestations of behavior and neurologicalfunction lags far behind. Difficulties in approaching these problemshave been caused, in part, by the large number of different cell typesin the mammalian CNS and the number and complexity of their connections.In addition, the blood-brain barrier makes access to the brain fordiagnosis, treatment and the design of new therapies more difficult.

In spite of the absence of sophisticated knowledge of thepathophysiology of most normal or abnormal brain functions, someattempts at pharmacological therapy for CNS dysfunction have alreadybecome useful and effective. These include the use of psycho-activedrugs for psychiatric disorders such as schizophrenia, and specificreplacement therapy in the rare cases in which the biochemical andcellular bases of the CNS disorder are relatively better understood, asin Parkinson's disease. At the core of most therapeutic approaches isthe objective of replacing or reactivating a specific chemical functionin the brain that has been lost as a consequence of disease or damage.

Intracerebral neural grafting has emerged recently as an additionalpotential approach to CNS therapy. The replacement or addition of cellsto the CNS which are able to produce and secrete therapeutically usefulmetabolites may offer the advantage of averting repeated drugadministration while also avoiding the drug delivery complications posedby the blood-brain barrier. (Rosenstein, Science 235: 772-774 (1987)).While the concepts and basic procedures of intracerebral grafting havebeen known for decades, most of the factors that optimize the survivalof grafted cells have only recently come to be investigated andpartially understood. (Bjorklund et al., in Neural Grafting in theMammalian CNS, p. 709, Elsevier, Amsterdam (1985); Sladek et al., inNeural Transplants: Development and Function, Plenum Press, N.Y.(1984)). Several factors critical for reliable and effective graftsurvival have been identified, including the following:

(1) Age of the donor: efficiency of grafting is reduced with increasingage of donor cells.

(2) Age of the host: young recipients accept grafts more readily thanolder ones.

(3) Availability of neurotrophic factors in the host and donor tissue:wound-induced neurotrophic factors enhance graft survival.

(4) Immunological response: the brain is not totally an immunologicallyprivileged site.

(5) The importance of target-donor matching: neurons survive better whenthey are grafted to a site which they would normally innervate.

(6) Vascularization: it is critical that the grafts be vascularizedrapidly or otherwise sufficiently well nourished from the environment.

As these critical factors have become recognized and optimized,intracerebral grafting has become a valid and reliable tool forneurobiologists in the study of CNS function and potentially forclinicians for the design of therapies of CNS disease. This approach hasreached a level of experimental clinical application in Parkinson'sdisease.

Parkinson's disease is an age-related disorder characterized by a lossof dopamine neurons in the substantia nigra of the midbrain, which havethe basal ganglia as their major target organ. The symptoms includetremor, rigidity and ataxia. The disease is progressive but can betreated by replacement of dopamine through the administration ofpharmacological doses of the precursor for dopamine, L-dopa, (Marsden,Trends Neurosci. 9: 512 (1986); Vinken et al., in Handbook of ClinicalNeurology p. 185, Elsevier, Amsterdam (1986)), although with chronic useof pharmacotherapy the patients often become refractory to the continuedeffect of L-dopa. There are many suggested mechanisms for thedevelopment of the refractory state, but the simplest is that thepatients reach a threshold of cell loss, wherein the remaining cellscannot synthesize sufficient dopamine from the precursor.

Parkinson's disease is the first disease of the brain for whichtherapeutic intracerebral grafting has been used in humans. Severalattempts have been made to provide the neurotransmitter dopamine tocells of the diseased basal ganglia of Parkinson's patients byhomografting adrenal medullary cells to the brain of affected patients.(Backlund et al., J. Neurosurg. 62: 169-173 (1985); Madrazo et al., NewEng. J. Med. 316: 831-836 (1987)). The transplantation of other donorcells such as fetal brain cells from the substantia nigra, an area ofthe brain rich in dopamine-containing cell bodies and also the area ofthe brain most affected in Parkinson's disease, has been shown to beeffective in reversing the behavioral deficits induced by selectivedopaminergic neurotoxins. (Bjorklund et al., Ann. N.Y. Acad. Sci. 457:53-81 (1986); Dunnett et al., Trends Neurosci. 6: 266-270 (1983)). Theseexperiments suggest that synaptic connectivity may not be a requisitefor a functional graft and that it may be sufficient to have cellsconstitutively producing and secreting dopamine in the vicinity of thedefective cells.

With this background, it seems likely that Parkinson's disease is acandidate disease for the transplantation of genetically engineeredcells, because (1) the chemical deficit is well known (dopamine), (2)the human and rat genes for the rate-limiting enzyme in the productionof dopamine have been cloned (tyrosine hydroxylase), (3) the anatomicallocalization of the affected region has been identified (basal ganglia),and (4) synaptic connectivity does not appear to be required forcomplete functional restoration.

The recent demonstration of genetic components in a rapidly growing listof other CNS diseases, including Huntington's disease, (Gusella et al.,Nature 306: 234-238 (1983)) Alzheimer's disease, (Delabar et al.,Science, N.Y. 235, 1390-1392 (1987); Goldgaber et al., Science, N.Y.235: 877-880 (1987); St. George-Hyslop et al., Science, N.Y. 235:885-890 (1987); Tanzi et al., Science, N.Y. 235: 880-884 (1987));bipolar disease (Baron et al., Nature 326: 289-292 (1987));schizophrenia (Sherrington et al., Nature 336: 164-167 (1988) and manyother major human diseases, suggests that gene therapy is an usefulapproach to treat these CNS diseases.

In parallel to the progress in neurobiology during the past severaldecades, advances in an understanding of molecular biology and thedevelopment of sophisticated molecular genetic tools have provided newinsights into human disease in general. As a result, medical scientistsand geneticists have developed a profound understanding of many humandiseases at the biochemical and genetic levels. The normal and abnormalbiochemical features of many human genetic. diseases have becomeunderstood, the relevant genes have been isolated and characterized, andearly model systems have been developed for the introduction offunctional wild-type genes into mutant cells to correct a diseasephenotype. (Anderson, Science 226: 401-409 (1984)). The extension ofthis approach to whole animals, that is, the correction of a diseasephenotype in vivo through the use of the functional gene as apharmacologic agent, has come to be called "gene therapy". (Friedmann etal., Science 175: 949-955 (1972); Friedmann, Gene Therapy Fact andFiction, Cold Spring Harbor Laboratory, N.Y. (1983)). Gene therapy isbased on the assumption that the correction of a disease phenotype canbe accomplished either by modification of the expression of a residentmutant gene or the introduction of new genetic information intodefective or damaged cells or organs in vivo.

At present, techniques for the ideal versions of gene therapy, that isthrough site-specific gene sequence correction or replacement in vivoare just beginning to be conceived but are not yet well developed.Therefore, most present models of gene therapy are actually geneticaugmentation rather than replacement models and rely on the developmentof efficient gene-transfer systems to introduce functional, wild-typegenetic information into genetically defective cells in vitro and invivo. To be clinically useful, the availability of efficient deliveryvectors for functional DNA sequences (transgenes) must be combined witheasy accessibility of suitable disease-related target cells or organsand with the development of techniques to introduce the vector stablyand safely into those target cells.

Model systems for the genetic and phenotypic correction of simpleenzymatic deficits are now being developed and studied, as is theidentification of the appropriate potential recipient cells and organsassociated with specific metabolic and genetic diseases. Evidence hasrecently been obtained to show that foreign genes introduced intofertilized mouse eggs can correct disease phenotype. (Constantini etal., Science 233: 1192-1394 (1986); Mason et al., Science 234: 1372-1378(1986); and Readhead et al., Cell 48: 703-712 (1987)).

A great deal of attention has recently been paid to the use of genedelivery vectors derived from murine retroviruses (Anderson, Science226: 401-409 (1984); Gilboa et al, Biotechniques 4: 504-512 (1986)) forgene transfer. Gene transfer in vitro using such retroviral vectors isextremely efficient for a broad range of recipient cells, the vectorshave a suitably large capacity for added genes, and infection with themdoes little metabolic or genetic damage to recipient cells. (Shimotohnoet al., Cell 26: 67-77 (1981); Wei et al., J. Virol. 39: 935-944 (1981);Tabin et al., Molec. Cell. Biol. 2: 426-436 (1982)). Several usefulsystems have demonstrated that the expression of genes introduced intocells by means of retroviral vectors can correct metabolic aberrationsin vitro in several human genetic diseases associated with single-geneenzyme deficiencies. (Willis et al., J. Biol. Chem. 259: 7842-7849(1984); Kantoff et al., Proc. Natl. Acad. Sci. USA 83: 6563-6567(1986)). There has been particular interest in bone marrow as apotential target organ for this approach to gene therapy because of theprevalence and importance of disorders of bone marrow-derived celllineages in a variety of major human diseases, including thethalassemias and sickle-cell anemia, Gaucher's disease, chronicgranulomatous disease (CGD) and immunodeficiency disease resulting fromdeficiencies of the purine pathway enzymes, adenosine deaminase (ADA)and purine nucleoside phosphorylase (PNP) (Kantoff, supra; McIvor etal., Molec. Cell. Biol. 7: 838-846 (1987); Soriano et al., Science 234:1409-1413 (1986); Willis et al., supra)). Other metabolically importanttarget organs, such as the liver, have also recently becometheoretically susceptible for genetic manipulation through thedemonstration of infection of cells from such organs with viral vectors(Wolff et al., Proc. Natl. Acad. Sci. USA 84: 3344-3348 (1987)).Furthermore, the discovery of numerous cell-specific regulatory signalssuch as cis-acting enhancers, tissue-specific promoters and othersequences may provide tissue specific gene expression in many otherorgans even after general, non-specific infections and gene transfer invivo (Khoury et al., Cell 33: 313-314 (1983); Serflin et al., TrendsGenet. 1: 224-230 (1985)).

A recently developed model of gene therapy uses target cells removedfrom a subject, placed in culture, genetically modified in vitro, andthen re-implanted into the subject (Wolff et al., Rheumatic Dis. Clin.N. Amer. 14(2) 459-477 (1988); Eglitis et al. Biotechniques 6: 608-614(1988)). Target cells have included bone marrow stem cells (Joyner etal., Nature 305: 556-558 (1983); Miller et al., Science 225: 630-632(1984); Williams et al., Nature 310: 476-480 (1984)); fibroblasts(Selden et al., Science 236: 714-718 (1982); Garver et al., Proc. Natl.Acad. Sci. USA 84: 1050-1054 (1987) and St. Louis et al., Proc. Natl.Acad. Sci. USA 85: 3150-3154 (1988)), keratinocytes (Morgan et al.,Science 237: 1476-1479 (1987)) and hepatocytes (Wolff et al., Proc.Natl. Acad. Sci. USA 84: 3344-3348 (1987)). This indirect approach of invivo gene transfer is necessitated by the inability to transfer genesefficiently directly into cells in vivo. Although there has been somerecent progress towards genetically modifying neurons in culture (Gelleret al., Science 241: 1667-1669 (1988)), this indirect approach of invivo gene transfer has not yet been applied to the CNS.

There are several ways to introduce a new function into target cells inthe CNS in a phenotypically useful way i.e. to treat defects, disease ordysfunction (FIG. 1). The most direct approach, which bypasses the needfor cellular grafting entirely, is the introduction of a transgenedirectly into the cells in which that function is aberrant as aconsequence of a developmental or genetic defect, i.e. neuronal cells inthe case of Tay-Sachs disease, possibly Lesch-Nyhan disease, andParkinson's disease (1, in FIG. 1). Alternatively, a new function isexpressed in defective target cells by introducing a geneticallymodified donor cell that could establish gap junction or other contactswith the target cell (2, in FIG. 1). Some such contacts are known topermit the efficient diffusion of metabolically important smallmolecules from one cell to another, leading to phenotypic changes in therecipient cell (Lowenstein, Biochim. Biophys. Acta. 560: 1-66 (1979)).This process has been called "metabolic cooperation" and is known tooccur between fibroblasts and glial cells (Gruber et al., Proc. Natl.Acad. Sci. USA 82: 6662-6666 (1985)), although it has not yet beendemonstrated conclusively in neurons. Still other donor cells couldexpress and secrete a diffusible gene product that can be taken up andused by nearby defective target cells (3, in FIG. 1). The donor cellsmay be genetically modified in vitro or alternatively they may bedirectly infected in vivo (4, in FIG. 1). This type of "co-operativity"has been demonstrated with CNS cells, as in the case of NGF-mediatedprotection of cholinergic neuronal death following CNS damage (Hefti, J.Neurosci. 6: 2155 (1986); Williams et al., Proc. Natl. Acad. Sci. USA83: 9231-9235 (1986)). Finally, an introduced donor cell infected withnot only replication-defective vector but also replication-competenthelper virus, could produce locally high titers of progeny virus thatmight in turn infect nearby target cells to provide a functional newtransgene (5, in FIG. 1).

There are several types of neurons in the mammalian brain. Cholinergicneurons are found within the mammalian brain and project from the medialseptum and vertical limb of the diagonal band of Broca to thehippocampal formation in the basal forebrain. The short, nerve-likeportion of the brain connecting the medial septum and vertical limb ofthe diagonal band with the hippocampal formation is termed the "fimbriafornix". The fimbria fornix contains the axons of the neurons located inthe medial septum and diagonal band. An accepted model of neuronsurvival in vivo is the survival of septal cholinergic neurons afterfimbria fornix transection or lesion (also termed "axotomy"). Axotomysevers the cholinergic neurons in the septum and diagonal band andresults in the death of up to one-half of the cholinergic neurons (Gageet al., Neuroscience 19: 241-256 (1986)). This degenerative response isattributed to the loss of trophic support from nerve growth factor(NGF), which is normally transported retrogradely in the intact brainfrom the hippocampus to the septal cholinergic cell bodies (Korsching etal., Proc. Natl. Acad. Sci. USA 80: 3513 (1983); Whittemore et al.,Proc. Natl. Acad. Sci. USA 83: 817 (1986); Shelton et al., Proc. Natl.Acad. Sci. USA 83: 2714 (1986); Larkfors et aI., J. Neurosci. Res. 18:525 (1987); and Seilor et al., Brain Res. 300: 33 (1984)).

Studies have shown that chronic intra-ventricular administration of NGFbefore axotomy will prevent cholinergic neuron death in the septum(Hefti, J. Neurosci. 8: 2155-2162 (1986); Williams et al., Proc. Natl.Acad. Sci. USA 83: 9231 (1986); Kromer, Science 235: 214 (1987); Gage etal. J. Comp. Neurol. 269: 147 (1988)). Axotomy thus provides an in vivomodel for determining at various points in time the ability of varioustherapies to prevent retrograde neuronal death.

One of the characteristics of the adult mammalian CNS is that new axonsgenerated following perturbation can only grow a relatively shortdistance within the brain (Cajal, in Degeneration and Regeneration ofthe Nervous System, Oxford University Press, London, England, (1928)).This inability of adult neurons to regenerate in response to damage maybe due to the following: 1) Activation of astrocytes, which are supportand nutritive cells found throughout the CNS, following injury resultsin the formation of scar tissue which acts as a physical barrier toregenerating axons such that fibers are unable to traverse the scartissue; thus astrocytic scars are unable to provide a conducivesubstrate for axon elongation (Cajal, supra; Brown, J. Comp. Neurol. 87:131 (1947); Clemente, in Regeneration in the Central Nervous System, ed.Windle, pp. 147-161, Thomas, Springfield, (1955); Windle, Physiol. Rev.36: 426 (1956); and Reier et al., in Spinal Cord Reconstruction, eds.Kao et al., pp. 163-195, Raven, New York, (1983)). Astrocytes in theimmature CNS, on the other hand, play an important role in the guidanceof elongating axons (Silver et al., J. Comp. Neurol. 210: 10-29 (1982));2) Myelin-associated substances released following damage to the brainhave been shown to inhibit axon growth in vitro (Schwab and Croni, J.Neurosci. 8: 2381-2393 (1988)) ; 3) The lack of axonal regeneration inthe adult CNS may be due, in part, to inadequate levels of trophic ortropic molecules which induce neuronal regeneration and promote axongrowth, respectively (Reier et al., in Neural Regeneration andTransplantation, pp. 183-209, Alan R. Liss, New York, (1989) andSchwartz et al., FASEB J. 3: 2371-2378 (1989)).

Despite many factors which may impede axon regrowth within the adultbrain, certain neurons in the rat CNS, especially retinal ganglionneurons and septal cholinergic neurons, exhibit a remarkable potentialto extend new axons into substrates of various types, so-called "neuralbridges", including segments of autologous peripheral nerve (David andAguayo, Science 214: 931 (1981); Benfry and Aguayo, Nature 296: 150(1982); Wendt et al., Exp. Neurol. 79: 452 (1983); and Hagg et al., Exp.Neurol. 109: 153 (1990)), cultured Schwann cells within a collagenmatrix (Kromer and Cornbrooks, Proc. Natl. Acad. Sci. USA 82: 6330(1985)), embryonic rat hippocampus (Kromer et al., Brain Res. 210: 153(1981)), and human amnionic membrane (Gage et al., Exp. Brain Res. 72:371 (1988)). Connectivity between the septum and hippocampus of thebrain has also been demonstrated using implants of peripheralhomogenates of neurons (Wendt, Brain Res. Bull. 15: 13-18 (1985)).

It would be advantageous, therefore, to develop procedures for genetransfer via efficient vectors followed by intracerebral grafting of thegenetically modified cells in vivo so as to ameliorate nerve celldisease, defect or dysfunction, and to promote axonal regeneration, totreat disorders of the CNS, such as Alzheimer's disease, Parkinson'sdisease and Huntington's disease.

SUMMARY OF THE INVENTION

The present invention provides methods for treating defective, diseasedor damaged cells in the mammalian central nervous system by graftinggenetically modified donor cells into the central nervous system toproduce functional molecules in a sufficient amount to ameliorate thedefective, diseased or damaged cells in the central nervous system. Thecells may be modified using viral or retroviral vectors, includingpLN.8RNL and pLThRNL, containing an inserted therapeutic transgene ortransgenes encoding a product or products which directly or indirectlyaffect the cells, or by other methods of introducing functional DNA intoa cell. The cells may be cultured and injected in suspension into thecentral nervous system and may be co-administered with a therapeuticagent for treating defective, diseased or damaged cells in the centralnervous system. The methods of the invention include selection,preparation and transfection of donor cells, and grafting.

The donor cells may be a mixture of cell types from different anatomicalregions and may be primary or immortalized cells.

Grafting of the donor cells may be accomplished by multiple grafting ofthe donor cells into several different sites within the mammalian CNS.For multiple grafting the cells may be a mixture of different types ofdonor cells, or may be the same or different types of cells containingthe same or different transgenes.

Grafting may be accompanied by implanting the cells in a substratematerial such as a collagen matrix to promote transplant survival and/orby the use of such material to facilitate reconnection between orameliorative interactions of injured neurons.

Vectors for use in the invention include those carrying a promoter suchas a collagen promoter to enhance expression of the transgene ortransgenes. The vectors may also carry enhancer sequences to increasethe activity of the promoter.

The secretion of the functional molecules may be regulated using aprecursor for said molecules. A method of the invention is the graftingof fibroblasts modified to express acetylcholine into the CNS and theadministration of choline chloride orally to maintain and enhance thesecretion of acetylcholine from the grafted fibroblasts.

The invention includes a method for enhancing the expression of atherapeutic transgene product from quiescent donor cells grafted into amammalian central nervous system, by inserting a quiescent promoter intoa vector used to genetically modify the donor cells. The promoter may bea non-viral promoter such as a collagen promoter. Additional enhancersequences may be inserted with the promoter. Cytokines may beadministered to regulate promoter activity to enhance transgeneexpression.

Immunosuppression agents may be used to reduce the production ofInterferon-γ to enhance transgene expression. Alternatively, ananti-inflammatory agent may be used to reduce the production ofcytokines to enhance the expression of therapeutic transgenes. Growthfactors may also be used to maintain the survival of the grafted donorcells in the CNS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of methods for introducing andanalyzing the effect of a new function in target cells.

FIG. 2 is a diagrammatic representation of strategies for introducing anew function into target cells in the CNS using genetically modifieddonor cells.

FIG. 3 is a diagrammatic depiction of the preparation of transmissibleretrovirus vectors containing a transgene. (GAG=group specific antigen;Env=envelope; POL=reverse transcriptase).

FIG. 4 is a diagrammatic representation of the linear restriction mapsof the integrated vectors LSΔPΔLM and pLNHL₂ as described in Example I,infra (arrows indicate the location of the promoter and the direction oftranscription. The diagonally hatched box of IS P LM represents thehuman HPRT cDNA encoding a protein with a novel terminal hexapeptideadded by in vitro mutagenesis, LTR=long terminal repeat).

FIG. 5 is a depiction of the circular restriction map of vector pLNHL₂as described in Example I, infra.

FIG. 6a-FIG. 6f are photomicrographs of primary rat fibroblastspreviously infected with hypoxanthine guanine phosphoribosyl transferase(HPRT) that have been implanted in rat basal ganglia as described inExample I, infra (FIG. 6a, FIG. 6d=anti- fibronectin; FIG. 6b, FIG.6e=cresyl violet; FIG. 6c, FIG. 6f=GFAP; magnification: FIG. 6a-FIG.6c=88X; FIG. 6d-FIG. 6f=440X).

FIG. 7 is photographs of isoelectric focusing gels for HPRT enzymaticactivity of brain extracts from basal ganglia as described in Example I,infra.

FIG. 8 is a depiction of the circular restriction map of vector pLLRNLas described in Example II, infra.

FIG. 9 is a depiction of the circular restriction map of vector pPR1 asdescribed in Example II, infra.

FIG. 10 is a depiction of the circular restriction map of vector pUCRHas described in Example II, infra.

FIG. 11 is a diagrammatic depiction of the linear restriction map of theintegrated NGF retroviral vector PLN.8RNL containing the 777 base pairHgal-Pstl fragment of mouse nerve growth factor (NGF) cDNA under controlof the viral 5' long terminal repeat (LTR) as described in Example II,infra (arrows indicate transcription initiation sites; psi (Ψ)=retroviral packaging signal; RSV=Rous sarcoma virus promoter; neo^(R)=neomycin-resistance gene marker.)

FIG. 12 is a depiction of the circular restriction map of vectorpLN.8RNL as shown in FIG. 11 and described in Example II, infra.

FIG. 13a-FIG. 13f are photomicrographs of immunohistochemical stainingfor fibronectin and ChAT as described in Example II, infra (FIG. 13a,FIG. 13b=fibronectin staining in fibroblasts grafted into the fimbriafornix cavity; FIG. 13c-FIG. 13f=coronal sections taken through themedial septum of tissue stained for ChAT; FIG. 13a, FIG. 13c, FIG.13e=animal with graft of retrovirus-infected cells; FIG. 13b, FIG. 13d,FIG. 13f=animal with graft of control cells; Magnification (Mag.): FIG.13a and 13b=20X; FIG. 13c and FIG. 13d=70 X; FIG. 13e and FIG.13f=220X).

FIG. 14 is a graph showing survival of ChAT-immunoreactive cells in theseptum of a rat in the presence and absence of NGF as described inExample II, infra.

FIG. 15a-FIG. 15f are photomicrographs of acetylcholinesterasehistochemistry as described in Example II, infra (FIG. 15a=low powermagnification of an animal grafted with NGF-infected donor cells; FIG.15c=higher power magnification of FIG. 15a through the medial septum;FIG. 15e=high power magnification of FIG. 15a through the dorsal lateralquadrant of the septum; FIG. 15b, FIG. 15d, FIG. 15f=animal grafted withcontrol cells as described for FIG. 15a, FIG. 15c, FIG. 15e; Mag.: FIG.15a, FIG. 15b=20X; FIG. 15c-FIG. 15f=220X).

FIG. 16 is a diagrammatic depiction of the linear restriction map ofpLThRNL retroviral integrated vector as described in Example III, infra(arrows indicate the location of the promoters and the direction oftranscription; LTR=long terminal repeat; RSV=modified RSV promoter;neo^(R) =neomycin-resistance gene).

FIG. 17 is a depiction of the derivation of vector pLThRNL as shown inFIG. 16 and described in Example II, infra.

FIG. 18a-FIG. 18d are photomicrographs of fibroblast grafts to thecaudate showing fibronectin immunoreactivity as described in ExampleIII, infra (Mag. FIG. 18a, FIG. 18c=10X; FIG. 18b, FIG. 18d=20X).

FIG. 19 is a graph showing the average percent change in the number ofrotations from baseline to post-transplantation in 4 experimental groupsof animals as described in Example III, infra (Top of figure: placementof control and TH-infected grafts in caudal striatum; Bottom: placementof control and TH-infected grafts in rostral striatum; bars indicatestandard deviation).

FIG. 20 is a graft of growth curves of primary skin fibroblasts andRat-1 fibroblasts in vitro as described in Example IV, infra.

FIG. 21 are histograms showing the volume (mm³) of autologous fibroblastgrafts after one and four passages in culture prior to grafting andafter three and eight weeks post-operative survival, as described inExample IV, infra.

FIG. 22 are histograms showing the volume (mm³) of autologous fibroblastgrafts resulting from implantations of 10⁴, 2.5×10⁴, 5 X 10⁴ and 10⁵cells/μl as described in Example IV, infra.

FIG. 23a-FIG. 23d are photographs showing primary fibroblast grafts (G)in the striatum stained immunohistochemically for fibronectin at three(FIG. 23a, FIG. 23c) and eight (FIG. 23b, FIG. 23d) weeks afterimplantation as described in Example IV, infra (Mag. FIG. 23a, FIG.23b=12X; FIG. 23c, FIG. 23d=20x).

FIG. 24a-FIG. 24f are photographs of primary fibroblast grafts (G) inthe striatum stained with cresyl violet (FIG. 24a, FIG. 24b) and stainedimmunohistochemically for GFAP (FIG. 24c, FIG. 24d) and OX-42 (FIG. 24e,FIG. 24f), at three weeks (FIG. 24a, FIG. 24c, FIG. 24e) and eight weeks(FIG. 24b, FIG. 24d, FIG. 24f) as described in Example IV, infra(Mag.=120X).

FIG. 25a-FIG. 25c are photographs showing the ultrastructure ofautologous grafts at eight weeks post implantation (F=fibroblast cellbodies; solid stars=fibroblast processes), as described in Example IV,infra (Mag. FIG. 25a=6,600X; FIG. 25b=9,100X; FIG. 25c=16,600X).

FIG. 26a14 FIG. 26b are photographs showing astrocytic processes andphagocytic cells present in the grafts (As=astrocytic processes) asdescribed in Example IV, infra (Mag. FIG. 26a=16,600X; FIG.26b=15,000X).

FIG. 27a-FIG. 27d are photographs showing graft vasculature as describedin Example IV, infra (FIG. 27a, E=non-fenestrated endothelial cells;FIG. 27b, arrowheads=invaginations; FIG. 27c, arrows=intercellularjunctions;

FIG. 27d, As=astrocytic processes; Mag. FIG. 27a=13,200X; FIG.27b=25,400X; FIG. 27c=25,000X; FIG. 27d=16,600X).

FIG. 28 is a photograph showing the results of in vitro labelling fortyrosine hydroxylase of primary fibroblasts containing a TH transgene asdescribed in Example V, infra (Mag.=25X).

FIG. 29 are graphs showing the results of grafts on apomorphine-inducedrotational behavior of 6-OHDA-lesioned rats as described in Example V,infra (Control, top; βGal fibroblast grafts, FF2/βGal, middle; THfibroblasts, FF2/TH, bottom; PRE=prior to grafting, **=p<0.01,*=p<0.05).

FIG. 30a14 FIG. 30b are photomicrographs showing the results of in situhybridization to TH primary fibroblasts grafted into the striatum ofrats as described in Example V, infra (FIG. 30a graft of FF2/TH cells;30b graft of FF2/βGal cells (control); G=graft; V=ventricle)).

FIG. 31a-FIG. 31d are photographs showing the results of in vivolabelling of grafted cells for either βGal histochemistry or in situhybridization to TH mRNA as described in Example V, infra (FIG. 31aFF2/βGal graft stained for βGal histochemistry; FIG. 31b highermagnification of FF2/βGal graft; FIG. 31c in situ hybridization for THmRNA in FF2/TH graft, arrow=fibroblasts; FIG. 31d in situ hybridizationto TH mRNA in FF2/βGal graft; Mag. FIG. 31a=10X; FIG. 31b, FIG. 31c andFIG. 31d=40X).

FIG. 32a-FIG. 32b are photographs showing the TH-immunoreactivity ofFF2/TH fibroblasts in vivo as described in Example V, infra(asterisks=rhodamine fluorescence; Mag.=20X).

FIG. 33 is a graph depicting the secretion of human NGF from genes andcell types as indicated, as described in Example VI, infra.

FIG. 34 is a schematic representation of the pLChRNL vector as describedin Example VII, infra.

FIG. 35a-FIG. 35f are photographs of immunostained fibroblasts asdescribed in Example VII, infra.

FIG. 36a-FIG. 36c illustrate the results of: Northern blot analysis oftotal RNA from fibroblasts (36a); assay of ChAT activity in fibroblasts(36b) and HPLC analysis of ACh secreted into the medium by fibroblasts(36c), as described in Example VII, infra.

FIG. 37a-FIG. 37b are graphs depicting the results of adding cholinechloride to cultures of Rat-1/dChAT cells as determined by HPLC anddescribed in Example VII, infra (FIG. 37a=effects on intracellular andextracellular ACh; FIG. 37b=effects on ChAT activity).

FIG. 38a-FIG. 38c illustrate the results of quiescence induced by serumstarvation on the expression of dChAT as shown by the effects on: ChATactivity (FIG. 38a); steady state levels of dChAT mRNA (FIG. 38b) and onthe ratio of dChAT/cyclophilin (cyc.) mRNA (FIG. 38c), as described inExample VII, infra.

FIG. 39a-FIG. 39c illustrate the effects of quiescence induced bycontact inhibition on the expression of dChAT as shown by the effectson: ChAT activity (FIG. 39a); levels of dChAT mRNA (FIG. 39b); and onthe ratio of dChAT/cyc. (FIG. 39c), as described in Example VII, infra.

FIG. 40 is a bar graph showing the effect of choline on the release ofACh from confluent fibroblasts as described in Example VII, infra.

FIG. 41 is a depiction of the circular restriction map of vectorpSVS32ACAT, as described in Example VIII, infra.

FIG. 42 is a depiction of the circular restriction map of vectorpMLVCAT, as described in Example VIII, infra.

FIG. 43 is a depiction of the circular restriction map of vectorpCMVCAT, as described in Example VIII, infra.

FIG. 44 is a bar graph depicting the relative CAT expression of the SV40, LTR and Collagen promoters (Coll and Coll(E) promoter-enhancer) asdescribed in Example VIII, infra.

FIG. 45a-FIG. 45b are photographs of Northern blots showing the effectsof TGFβ, IL-1β and TNFα on dChAT-expressing fibroblasts, as described inExample IX, infra (FIG. 45a: effect of TGF-β (lane 2) and IL-1β (lane3); FIG. 45b: effect of Infγ, lane 1=control; cyclophilin (Cyc)=internalcontrol; lane 2=effects after 24 hrs; lane 3=effects after 48 hrs;dChAT=dChAT proviral mRNA).

FIG. 46 is a photograph of a Northern blot showing the effects ofcoadministration of TGFβ and IL-1β, and dexamethasone, on confluentdChAT-producing fibroblasts, as described in Example IX, infra (lane1=control; lane 2=TGFβ/IL-1β; lane 3=dexamethasone alone; lane4=TGFβ/IL-1β and dexamethasone; top signals=dChAT mRNA; lowersignals=cyclophilin mRNA).

FIG. 47a-FIG. 47b are bar graphs showing the activity of the collagenpromoter in 10% versus 2% serum (FIG. 47a) and the effect of variouscytokines on the collagen promoter (FIG. 47b) as described in ExampleIX, infra.

FIG. 48a-FIG. 48d are photomicrographs showing grafts of LTR-CAT (FIG.48a, FIG. 48b) or Coll(E)-CAT (FIG. 48c, FIG. 48d) cells grafted intothe striatum of adult Fischer rats as described in Example IX, infra(white arrows=fibroblasts).

FIG. 49a-FIG. 49d are photomicrographs showing horizontal sectionsthrough the left and right striatum at one (FIG. 49a, FIG. 49b) andeight (FIG. 49c, FIG. 49d) weeks after implants (genetically modifiedcells injected into the right striatum and non-infected controlsinjected into the left striatum) as described in Example X, infra(G=grafts; NG=nucleus basalis of Meynert; RT=reticular thalamic nucleus;scale bars: FIG. 49a, FIG. 49b, 0.45 mm; FIG. 49c, FIG. 49d, 0.22 mm).

FIG. 50 is an electron photomicrograph of a graft composed ofNGF-producing cells eight weeks post-implantation as described inExample X, infra (F=fibroblasts; E=endothelial cells; arrow=astrocyticprocesses; scale bar=1.0 μm).

FIG. 51 is a photomicrograph and schematic illustration of an axo-glialarrangement within a graft of NGF-producing cells at eight weeks asdescribed in Example X, infra (Ax=unmyelinated axons; As=reactiveastrocytic processes; arrowheads=basal lamina; scale bar=1.0 μm).

FIG. 52 is a schematic illustration of a sagittal view of the adult ratforebrain after unilateral fimbria-fornix (FF) ablation and placement ofa graft consisting of collagen with either NGF-producing or control,non-infected primary fibroblasts, as described in Example X, infra(MS=media septum, G=graft, and DG=hippocampal dentate gyrus. FIG. 52a,FIG. 52b show coronal tissue sections at comparable levels through themedial septum (see plane indicated by "A,B" in schematic above) stainedimmunohistochemically for NGF receptor after unilateral FF aspirativelesion and placement of either NGF-producing (FIG. 52a) or control (FIG.52b) grafts (arrowheads indicate septal midline); FIG. 52c, FIG. 52dshow coronal sections through grafts (see plane indicated by "C,D" inschematic above) of either NGF-producing (FIG. 52c) or control,non-infected (FIG. 52d) fibroblasts stained for AChE (dotted linerepresents approximate boundary of the graft, scale bars in FIG. 52b and52c=100 μm; scale bar in FIG. 52d=200 μm); FIG. 52e, FIG. 52f, FIG.52gshow coronal sections through the hippocampal dentate gyrus (seeplane indicated by "E,F" in schematic above) stained for AChE(G=granular layer;, IM=inner molecular layer; OM=outer molecular layer,and P=polymorphic layer)).

FIG. 53a-FIG. 53d are photomicrographs showing retrogradelyfluorescently labeled septal neurons following stereotaxic placement offluorescent microspheres in the dentate gyrus after surgery in animalsthat received NGF-producing grafts as described in Example X, infra(FIG. 53a=ipsilateral medial septum; FIG. 53b=contralateral diagonalband; FIG. 53c=septal midline; and FIG. 53d=ipsilateral diagonal band;scale bar=50 μm).

FIG. 54a-FIG. 54f are electron photomicrographs showing theultrastructural distribution of unmyelinated axons within grafts ofcollagen and NGF-producing fibroblasts after surgery as described inExample X, infra (*=astrocytic processes; S=Schwann cells; L=lumen ofthe capillary; scale bars=1.0 μm).

FIG. 55a-FIG. 55f depicts topographical (FIG. 55a) and synaptic (FIG.55b-f) distribution of AChE activity within the dentate gyrus after FFablation and implants of NGF-producing fibroblasts in a collagen matrixas described in Example X, infra (FIG. 55a=coronal section (40 μm) ofthe dentate gyrus stained for ACHE, taken immediately adjacent to thattissue examined at the ultrastructural level, IM=inner molecular layer,OM=outer molecular layer; G=granular layer; P=polymorphic layer; FIG.55b=granular layer; FIG. 55c=molecular layer; FIG. 55d,e,f,arrowheads=synaptic contacts, Sh=dendritic shafts; Sp=spines,As=astrocytic processes, scale bars: in FIG. 55a=25 μm; in FIG. 55b=0.5μm, in FIG. 55c and representative for FIG. 55d-FIG. 55f=0.25 μm).

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth.

The present invention relates to a process for grafting geneticallymodified donor cells into the mammalian central nervous system (CNS) totreat disease or trauma of the CNS. More particularly, the inventionrelates to the use of vectors carrying functional gene inserts(transgenes) to modify donor cells to produce molecules that are capableof directly or indirectly affecting cells in the CNS to repair damagesustained by the cells from defects, disease or trauma. Preferably, fortreating defects, disease or damage of cells in the CNS, donor cellssuch as fibroblasts are modified by introduction of a retroviral vectorcontaining a transgene or transgenes, for example a gene encoding nervegrowth factor (NGF) protein. The genetically modified fibroblasts aregrafted into the central nervous system, for example the brain, to treatdefects, disease such as Alzheimer's or Parkinson's, or injury fromphysical trauma, by restoration or recovery of function in the injuredneurons as a result of production of the expressed transgene product(s)from the genetically modified donor cells. The donor cells may also beused to introduce a transgene product or products into the CNS thatenhance the production of endogenous molecules that have ameliorativeeffects in vivo.

As used herein the term "transgene" or "therapeutic transgene" means DNAinserted into a donor cell encoding an amino acid sequence correspondingto a functional protein capable of exerting a therapeutic effect oncells of the CNS or having a regulatory effect on the expression of afunction in the cells of the CNS.

Gene Transfer Into Donor Cells In Vitro

The strategy for transferring genes into donor cells in vitro isoutlined in FIG. 2 and includes the following basic steps: (1) selectionof an appropriate transgene or transgenes whose expression is correlatedwith CNS disease or dysfunction; (2) selection and development ofsuitable and efficient vectors for gene transfer; (3) preparation ofdonor cells from primary cultures or from established cell lines; (4)demonstration that the donor implanted cells expressing the new functionare viable and can express the transgene product(s) stably andefficiently; (5) demonstration that the transplantation causes noserious deleterious effects; and (6) demonstration of a desiredphenotypic effect in the host animal.

The functional molecules produced by transgenes for use in the inventioninclude, but are not limited to, growth factors, enzymes, gangliosides,antibiotics, neurotransmitters, neurohormones, toxins, neurite promotingmolecules, antimetabolites and precursors of these molecules. Inparticular, transgenes for insertion into donor cells include, but arenot limited to, tyrosine hydroxylase, tryptophan hydroxylase, NGF, ChAT,GABA-decarbokylase, Dopa decarboxylase (AADC), enkephlin, ciliaryneuronal trophic factor (CNTF), brain derived neurotrophic factor(BDNF), neurotrophin (NT)-3, NT-4, and basic fibroblast growth factor(bFGF).

Genetic Modification of Donor Cells

The methods described below to modify donor cells using retroviralvectors and grafting into the CNS are merely for purposes ofillustration and are typical of those that might be used. However, otherprocedures may also be employed, as is understood in the art.

Most of the techniques used to transform cells, construct vectors andthe like are widely practiced in the art, and most practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures. However, for convenience, the followingparagraphs may serve as a guideline.

Choice of donor cells

The choice of donor cells for implantation depends heavily on the natureof the expressed gene, characteristics of the vector and the desiredphenotypic result. Because retroviral vectors require cell division andDNA synthesis for efficient infection, integration and gene expression(Weiss et al., RNA Tumor viruses, 2nd Ed., Weiss et al., eds., ColdSpring Harbor Press, N.Y. (1985)), if such vectors are used, the donorcells are preferably actively growing cells such as primary fibroblastculture or established cell lines, replicating embryonic neuronal cellsor replicating adult neuronal cells from selected areas such as theolfactory mucosa and possibly developing or reactive glia. Primarycells, i.e. cells that have been freshly obtained from a subject, suchas fibroblasts, that are not in the transformed state are preferred foruse in the present invention. Other suitable donor cells includeimmortalized (transformed cells that continue to divide) fibroblasts,glial cells, adrenal cells, hippocampal cells, keratinocytes,hepatocytes, connective tissue cells, ependymal cells, bone marrowcells, stem cells, leukocytes, chromaffin cells and other mammaliancells susceptible to genetic manipulation and grafting using the methodsof the present invention.

The application of methods to induce a state of susceptibility instationary, non-replicating target cells may make many other cell typessuitable targets for viral transduction. For instance, methods have beendeveloped that permit the successful retrovital vector infection ofprimary cultures of adult rat hepatocytes, ordinarily refractory toinfection with such vectors, and similar methods may be helpful for anumber of other cells (Wolff et al., Proc. Natl. Acad. Sci. USA 84:3344-3348 (1987)). In addition, the development of many other kinds ofvectors derived from herpes, vaccinia, adenovirus, or other viruses, aswell as the use of efficient non-viral methods for introducing DNA intodonor cells such as electroporation (Toneguzzo et al., Molec. Cell.Biol. 6: 703-706 (1986)), lipofection or direct gene insertion may beused for gene transfer into many other cells presently not susceptibleto retroviral vector infection.

Additional characteristics of donor cells which are relevant tosuccessful grafting include the age of the donor cells. The resultspresented herein demonstrate that aged human cells may be used fortransfection with transgenes for grafting.

Choice of Vector

Although other vectors may be used, preferred vectors for use in themethods of the present invention are viral, including retroviral,vectors. The viral vector selected should meet the followingcriteria: 1) the vector must be able to infect the donor cells and thusviral vectors having an appropriate host range must be selected; 2) thetransferred gene should be capable of persisting and being expressed ina cell for an extended period of time without causing cell death forstable maintenance and expression in the cell; and 3) the vector shoulddo little, if any, damage to target cells. Murine retroviral vectorsoffer an efficient, useful, and presently the best-characterized meansof introducing and expressing foreign genes efficiently in mammaliancells. These vectors have very broad host and cell type ranges,integrate by reasonably well understood mechanisms into random sites inthe host genome, express genes stably and efficiently, and under mostconditions do not kill or obviously damage their host cells.

General Methods for Vector Construction

Construction of suitable vectors containing the desired therapeutic genecoding and control sequences employs standard ligation and restrictiontechniques, which are well understood in the art (see Maniatis et al.,in Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. (1982)). Isolated plasmids, DNA sequences, orsynthesized oligonucleotides are cleaved, tailored, and religated in theform desired.

Site-specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes (See,e.g. New England Biolabs Product Catalog.) In general, about 1 μg ofplasmid or DNA sequences is cleaved by one unit of enzyme in about 20 μlof buffer solution. Typically, an excess of restriction enzyme is usedto insure complete digestion of the DNA substrate.

Incubation times of about one hour to two hours at about 37° C. areworkable, although variations can be tolerated. After each incubation,protein is removed by extraction with phenol/chloroform, and may befollowed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol. If desired, sizeseparation of the cleaved fragments may be performed by polyacrylamidegel or agarose gel electrophoresis using standard techniques. A generaldescription of size separations is found in Methods in Enzymology 65:499-560 (1980).

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 min at 20° C. to 25° C. in 50 mM Tris (pH 7.6) 50 mMNaCl,6 mM MgCl₂, 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fills in at5' sticky ends but chews back protruding 3' single strands, even thoughthe four dNTPs are present. If desired, selective repair can beperformed by supplying only one of the dNTPs, or with selected dNTPs,within the limitations dictated by the nature of the sticky ends. Aftertreatment with Klenow, the mixture is extracted with phenol/chloroformand ethanol precipitated. Treatment under appropriate conditions with S1nuclease or Bal-31 results in hydrolysis of any single-stranded portion.

Ligations are performed in 15-50 μl volumes under the following standardconditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 mg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP, 0.01-0.02(Weiss) units T4 DNA ligase at 15° C. (for "sticky end" ligation) or 1mMATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 25° C. (for "blunt end"ligation). Intermolecular "sticky end" ligations are usually performedat 33-100 μg/ml total DNA concentrations (5-100 nM total endconcentration). Intermolecular blunt end ligations (usually employing a10-30 fold molar excess of linkers) are performed at 1 μM total endsconcentration.

In vector construction employing "vector fragments", the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) or calfintestinal alkaline phosphatase (CIP) in order to remove the 5'phosphate and prevent religation of the vector. Digestions are conductedat pH 8 in approximately 150 mM Tris, in the presence of Na+and Mg+2using about 1 unit of BAP or CIP per mg of vector at 37° C. for aboutone hour. In order to recover the nucleic acid fragments, thepreparation is extracted with phenol/chloroform and ethanolprecipitated. Alternatively, religation can be prevented in vectorswhich have been double digested by additional restriction enzymedigestion of the unwanted fragments.

Methods of preparation of retroviral vectors have been described (Yee etal., Cold Spring Harbor Symp. on Quant. Biol. Vol. LI, pp. 1021-1026(1986); Wolff et al., Proc. Natl. Acad. Sci. USA 84: 3344-3348 (1987);Jolly et al., Meth. in Enzymol. 149: 10-25 (1987); Miller et al., Mol.Cell. Biol. 5: 431-437 (1985); and Miller, et al., Mol. Cell. Biol. 6:2895-2902 (1986) and Eglitis et al., Biotechniques 6: 608-614 (1988))and are now in common use in many laboratories. Retroviral vectorscontain retroviral long terminal repeats (LTRs) and packaging (psi, Ψ)sequences, as well as plasmid sequences for replication in bacteria andmay include other sequences such as the SV40 early promoter and enhancerfor potential replication in eukaryotic cells. Much of the rest of theviral genome is removed and replaced with other promoters and genes.Vectors are packaged as RNA in virus particles following transfection ofDNA constructs into packaging cell lines. These include psi (Ψ)2 whichproduce viral particles that can infect rodent cells and ΨAM and PA 12which produce particles that can infect a broad range of species.

In a preferred viral vector the transgene is brought under the controlof either the viral LTR promoter-enhancer signals or of an internalpromoter, and retained signals within the retroviral LTR can still bringabout efficient integration of the vector into the host cell genome. Toprepare transmissible virus (FIG. 3), recombinant DNA molecules of suchdefective vectors are transfected into "producer" cell lines thatcontain a provirus expressing all of the retroviral functions requiredfor packaging of viral transcripts into transmissible virus particles,but lacking the crucial packaging signal for encapsidation of RNAtranscripts of the provirus into mature virus particles. These includethe group specific antigen (GAG) and envelope (ENV) genes which encodecaps id proteins and reverse transcriptase (POL). Because of thisdeletion, transcripts from the helper cannot be packaged into viralparticles and the producer cells, therefore, generate only empty virusparticles. However, an integrated defective retroviral vector introducedinto the same cell by means of calcium phosphate-mediated transfection(Graham and Vander Eb, Virol. 52: 456-467 (1973)) in which the GAG, ENVand POL genes have been replaced by the transgene (x) with the intactpsi sequence, produces transcripts that can be packaged in trans sincethey do contain the packaging sequence. The cells contain two provirussequences integrated into different sites of the host cell genome.Because RNA transcripts from the newly introduced provirus contain thepackaging sequence they are efficiently encapsidated into virusparticles by means of viral functions produced in trans. Ideally, theresult is the production by the cells of infectious particles carryingthe transgene free of replication-competent wild-type helper virus. Inmost, but not necessarily all models of gene therapy, the production ofhelper virus is probably undesirable since it may lead to spreadinginfection and possibly proliferative disease in lymphoid or other tissuein the host animal.

Because herpes viruses are capable of establishing a latent infectionand an apparently non-pathogenic relationship with some neural cells,herpes based vectors, e.g. HSV-1, may be used. Similarly, it should bepossible to take advantage of an eventual improved understanding ofother human and animal viruses that infect cells of the CNS efficiently,such as rabies virus, measles, and other paramyxoviruses and the humanimmunodeficiency retrovirus (HIV), to develop useful delivery andexpression vectors. In most cases, with the exception of rabies virus,these viruses are not truly neurotropic for infection, but rather have amuch more general susceptible host cell range. They seem, rather, toappear to be neurotropic because the metabolic and physiological effectsof infection are most pronounced in cells of the CNS. It is, therefore,likely that many vectors derived from these viruses will be similarlypromiscuous in their cell range, and that CNS specificity for expressionmust be conferred by the use of appropriate cell-specific enhancer,promoter and other sequences, such as those that regulate theoligodendroglial-specific expression of JC virus, glial-specificexpression of the proteolipid protein and glial fibrillary acidicprotein (GFAP) genes, and other possible CNS specific functions in themouse.

Other virus vectors that may be used for gene transfer into cells forcorrection of CNS disorders include retroviruses such as Moloney murineleukemia virus (MoMuLV); papovaviruses such as JC, SV40, polyoma,adenoviruses; Epstein-Barr Virus (EBV); papilloma viruses, e.g. bovinepapilloma virus type I (BPV); vaccinia and poliovirus and other humanand animal viruses.

A possible problem posed by the use of defective vital vectors is thepotential for the eventual emergence or "rescue" of pathogenic,replication-competent, wild- type virus by recombination with-endogenousvirus-like or other cellular sequences. This possibility can be reducedthrough the elimination of all viral regulatory sequences not needed forthe infection, stabilization or expression of the vector.

In addition to the above-described methods for inserting functional DNAtransgenes into donor cells other methods may be used. For example,non-vector methods include nonviral physical transfection of DNA intocells; for example, microinjection (DePamphilis et al., BioTechnique 6:662-680 (1988)); electroporation (Tonequzzo et al., Molec. Cell. Biol.6: 703-706 (1986), Potter, Anal. Biochem. 174:: 361-373 (1988));chemically mediated transfection such as calcium phosphate transfection(Graham and van der EB, supra, Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987), Chen and Okayama, BioTechnique, 6: 632-638 (1988)) andDEAE-dextran mediated transfer (McCutchan and Pagano, J. Natl. CancerInst. 41: 351-357 (1968)); cationic liposomal mediated transfection(Felgner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987),Felgner and Holm, Focus 11: 21-25 (1989) and Felgner et al., Proc. West.Pharmacol. Soc. 32: 115-121 (1989)) and other methods known in the art.

Mechanisms of Phenotypic Correction by Donor Cells

Preparation of Donor Cells

The donor cells must be properly prepared for grafting. For example, forinjection of genetically modified donor cells according to the presentinvention, cells such as fibroblasts obtained from skin samples areplaced in a suitable culture medium for growth and maintenance of thecells, for example a solution containing fetal calf serum (FCS) andallowed to grow to confluency. The cells are loosened from the culturesubstrate for example using a buffered solution such as phosphatebuffered saline (PBS) containing 0.05% trypsin and placed in a bufferedsolution such as PBS supplemented with 1 mg/ml of glucose; 0.1 mg/ml ofMgCl₂ ; 0.1 mg/ml CaCl₂ (complete PBS) plus 5% serum to inactivatetrypsin. The cells may be washed with PBS using centrifugation and arethen resuspended in the complete PBS without trypsin and at a selecteddensity for injection. In addition to PBS, any osmotically balancedsolution which is physiologically compatible with the host subject maybe used to suspend and inject the donor cells into the host.

The long-term survival of implanted cells may depend on effects of theviral infection on the cells, on cellular damage produced by the cultureconditions, on the mechanics of cell implantation, or the establishmentof adequate vascularization, and on the immune response of the hostanimal to the foreign cells or to the introduced gene product. Themammalian brain has traditionally been considered to be animmunologically privileged organ, but recent work has shown conclusivelythat immune responses can be demonstrated to foreign antigens in the ratbrain. It is important to minimize the potential for rejection andgraft-versus-host reaction induced by the grafted cells by usingautologous cells wherever feasible, by the use of vectors that will notproduce changes in cell surface antigens other than those associatedwith the phenotypic correction and possibly by the introduction of thecells during a phase of immune tolerance of the host animal, as in fetallife.

The most effective mode and timing of grafting of the transgene donorcells of the invention to treat defects, disease or trauma in the CNS ofa patient will depend on the severity of the defect and on the severityand course of disease or injury to cells such as neurons in the CNS, thepatient's health and response to treatment and the judgment of thetreating health professional.

Of course, as in all other gene-transfer systems, the important issuesof appropriate or faithful gene expression must be resolved to ensurethat the level of gene expression is sufficient to achieve the desiredphenotypic effect and not so high as to be toxic to the cell.

A problem associated with the use of genetically engineered cells astransplants for gene therapy is that as cells become quiescent(non-dividing) the expression of genes, including transgenes, has beenobserved to decrease ("down regulate") (Palmer et al., Proc. Natl. Acad.Sci. USA 88: 1330-334 (1991)). Primary fibroblasts grafted into thebrain do not continue to divide when implanted unless they aretransformed and tumorigenic. They thus exist in a quiescent state in thebrain. It is thus useful to provide means for maintaining and/orincreasing expression of the transgene in the absence of cell divisionto promote long term stable expression of therapeutic genes used infibroblasts for gene therapy.

Expression of a gene is controlled at the transcription, translation orpost-translation levels. Transcription initiation is an early andcritical event in gene expression. This depends on the promoter andenhancer sequences and is influenced by specific cellular factors thatinteract with these sequences. The transcriptional unit of manyprokaryotic genes consists of the promoter and in some cases enhancer orregulator elements (Banerji et al., Cell 27: 299 (1981); Corden et al.,Science 209: 1406 (1980); and Breathnach and Chambon, Ann. Rev. Biochem.50: 349 (1981)). For retroviruses, control elements involved in thereplication of the retroviral genome reside in the long terminal repeat(LTR) (Weiss et al., eds., In: The molecular biology of tumor viruses:RNA tumor viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor,New York (1982)). Moloney murine leukemia virus (MLV) and Rous sarcomavirus (RSV) LTRs contain promoter and enhancer sequences (Jolly et al.,Nucleic Acids Res. 11: 1855 (1983); Capecchi et al., In: Enhancer andeukaryotic gene expression, Gulzman and Shenk, eds., pp. 101-102, ColdSpring Harbor Laboratories, Cold Spring Harbor, New York). Promoter andenhancer regions of a number of non-viral promoters have also beendescribed (Schmidt et al., Nature 314: 285 (1985); Rossi and deCrombrugghe, Proc. Natl. Acad. Sci. USA 84: 5590-5594 (1987)).

The present invention provides methods for maintaining and increasingexpression of transgenes in quiescent cells using promoters includingcollagen type I (α1 and α2) (Prockop and Kivirikko, N. Eng. J. Med. 311:376 (1984); Smith and Niles, Biochem. 19: 1820 (1980); de Wet et al., J.Biol. Chem. 258: 14385 (1983)), SV40 and LTR promoters.

In addition to using viral and non-viral promoters to drive transgeneexpression in donor cells such as primary fibroblasts, an enhancersequence may be used to increase the level of transgene expression.Enhancers can increase the transcriptional activity not only of theirnative gene but also of some foreign genes (Armelor, Proc. Natl. Acad.Sci. USA 70: 2702 (1973)). For example, in the present invention,collagen enhancer sequences are used with the collagen promoter α2(I) toincrease transgene expression. In addition, the enhancer element foundin SV40 viruses may be used to increase transgene expression. Thisenhancer sequence consists of a 72 basepair repeat as described by Grusset al., Proc. Natl. Acad. Sci. USA 78: 943 (1981); Benoist and Chambon,Nature 290: 304 (1981), and Fromm and Berg, J. Mol. Appl. Genetics, 1:457 (1982), all of which are incorporated by reference herein. Thisrepeat sequence can increase the transcription of many different viraland cellular genes when it is present in series with various promoters(Moreau et al., Nucleic Acids Res. 9: 6047 (1891)). In most cases, theSV40 enhancer element is fully functional when it is present in eitherorientation and/or at a variety of positions in the plasmid DNA (Frommand Berg; supra). The ability of the SV40 enhancer to uprequlate thetranscription from the α2(I) collagen promoter (Coll) has beenpreviously demonstrated in embryonic chicken fibroblasts (CEF) andmonkey kidney CV-1 cells (Xu et al., In Enhancer and Eukaryotic GeneExpression, Gulzman and Shenk, eds., Cold Spring Harbor Laboratories,Cold Spring Harbor, N.Y., pp. 51-54 (1983)). In these cells, vectorscontaining the SV40 enhancer region downstream from the α2(I) collagenpromoter increased chloramphenical acetyltransferase activity, thereporter gene used, by 64-fold and 18-fold in CV-1 and CEF cells,respectively.

Transgene expression may also be increased for long term stableexpression after grafting using cytokines to modulate promoter activity.Several cytokines have been reported to modulate the expression oftransgene from collagen α2(I) and LTR promoters (Chua et al., ConnectiveTissue Res. 25: 161-170 (1990); Elias et al., Annals N.Y. Acad. Sci.580: 233-244 (1990)); Seliger et al., J. Immunol. 141: 2138-2144 (1988)and Seliger et al., J. Virology 62: 619-621 (1988)). For example,transforming growth factor (TGF)β, interleukin (IL)-1β, and interferon(Inf)α or γ down regulate the expression of transgenes driven by variouspromoters such as LTR. Tumor necrosis factor (TNF)α and TGFβ1 upregulate and IL-1β up regulates the expression of transgenes driven bythe α2(I) collagen promoter (Coll). The results presented hereindemonstrate that some cytokines such as TNFα, TGFβ and IL-1β, may beused to control expression of transgenes driven by a promoter in donorcells such as fibroblasts. Other cytokines that may prove useful includebasic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).

Collagen promoter with the collagen enhancer sequence (Coil(E)) can alsobe used to take advantage of the high level of cytokines present in thebrain following grafting of the modified donor cells to increasetransgene expression. In addition, anti-inflammatory agents includingsteroids, for example dexamethasone, may be administered to the graftrecipient immediately after implantation of the fibroblasts andcontinued, preferably, until the cytokine-mediated inflammatory responsesubsides. In certain cases, an immunosuppression agent such ascyclosporin may be administered to reduce the production of interferon-γwhich downregulates LTR promoter and Coll(E) promoter-enhancer, andreduces transgene expression.

For in vivo use, transgenes driven by collagen promoter are introducedinto cells and then directly implanted into the brain without requiringfurther intervention. The cytokines released after grafting as part ofthe recipient's natural response will stimulate the collagen promoterdriven transcription of the selected transgene.

Cytokines including the growth factors bFGF and EGF, may also beadministered before, during or after grafting, to promote survival ofgrafted donor cells in the CNS.

It is also useful to be able to regulate the secretion of thegenetically engineered gene product after grafting. As shown in anembodiment presented herein, the release of a gene product such asacetylcholine (ACh), a transmitter, greatly decreased in Alzheimer'sDisease, from cultured cells infected with a MLV vector expressing thecholine acetyltransferase cDNA can be augmented using choline, aprecursor for acetylcholine. This suggests a means for dietaryregulation of intracerebral gene therapy.

The genetic correction of some, or many, CNS disorders may require theestablishment or re-establishment of faithful intercellular synapticconnections. Model systems to study these possibilities have not yetbeen developed and exploited because of the paucity of replicatingnon-transformed cell-culture systems and the refractoriness ofnon-replicating neuronal cells to retroviral infection. However, recentstudies, including those involving the immortalization of embryonichippocampal neuronal cells, suggest that replicating neuronal cellculture systems may soon become available for in vitro gene transfer andthen for in vivo implantation (Caettano and MacKay, Nature 347: 762-765(1990)). Such neurons might be susceptible to efficient transduction byretroviral or other viral vectors, and if they are also able to retainother neuronal characteristics, they may be able to establish synapticconnections with other cells after grafting into the brain.Alternatively, there are cells within the CNS that are late to develop,such as the ventral leaf of the dentate gyrus of the hippocampus, orcontinue to divide through adulthood, such as those in the olfactorymucosa and in the dentate gyrus. Such cells may be suitable targets forretroviral infection.

The use of non-neuronal cells for grafting may preclude the developmentof specific neural connections to resident target cells of the host.Therefore, the phenotypic effects of fibroblast or other non-neuronaldonor cells or target cells in vivo would be through the diffusion of arequired gene product or metabolite, through gap junctions ("metabolicco-operation") or through uptake by target cells of secreted donor cellgene products or metabolites. The donor cell may also act as a toxin"sink" by expressing a new gene product and metabolizing and clearing aneurotoxin.

Alternatively, neural bridges may be provided which facilitatereconnection between neurons in damaged CNS tissues. As noted above,grafted donor cells suspended in substrate material such as collagenmatrices can serve as neural bridges to facilitate axonal regenerationand reconnection of injured neurons, or may be used in conjunction withneural bridges formed from synthetic or biological materials, forexample homogenates of neurons or placenta, or neurite promotingextracellular matrices.

Grafting

The methods of the invention contemplate intracerebral grafting of donorcells containing a transgene insert to the region of the CNS havingsustained defect, disease or trauma.

Neural transplantation or "grafting" involves transplantation of cellsinto the central nervous system or into the ventricular cavities orsubdurally onto the surface of a host brain. Conditions for successfultransplantation include: 1) viability of the implant; 2) retention ofthe graft at the site of transplantation; and 3) minimum amount ofpathological reaction at the site of transplantation.

Methods for transplanting various nerve tissues, for example embryonicbrain tissue, into host brains have been described in Neural Grafting inthe Mammalian CNS, Bjorklund and Stenevi, eds., (1985) Das, Ch. 3 pp.23-30; Freed, Ch. 4, pp. 31-40; Stenevi et al., Ch. 5, pp. 41-50;Brundin et al., Ch. 6, pp. 51-60; David et al., Ch. 7, pp. 61-70;Seiger, Ch. 8, pp. 71-77 (1985), incorporated by reference herein. Theseprocedures include intraparenchymal transplantation, i.e. within thehost brain (as compared to outside the brain or extraparenchymaltransplantation) achieved by injection or deposition of tissue withinthe host brain so as to be opposed to the brain parenchyma at the timeof transplantation (Das, supra).

The two main procedures for intraparenchymal transplantation are: 1)injecting the donor cells within the host brain parenchyma or 2)preparing a cavity by surgical means to expose the host brain parenchymaand then depositing the graft into the cavity (Das, supra). Both methodsprovide parenchymal apposition between the graft and host brain tissueat the time of grafting, and both facilitate anatomical integrationbetween the graft and host brain tissue. This is of importance if it isrequired that the graft become an integral part of the host brain and tosurvive for the life of the host.

Alternatively, the graft may be placed in a ventricle, e.g. a cerebralventricle or subdurally, i.e. on the surface of the host brain where itis separated from the host brain parenchyma by the intervening pia materor arachnoid and pia mater. Grafting to the ventricle may beaccomplished by injection of the donor cells or by growing the cells ina substrate such as 3% collagen to form a plug of solid tissue which maythen be implanted into the ventricle to prevent dislocation of thegraft. For subdural grafting, the cells may be injected around thesurface of the brain after making a slit in the dura. Injections intoselected regions of the host brain may be made by drilling a hole andpiercing the dura to permit the needle of a microsyringe to be inserted.The microsyringe is preferably mounted in a stereotaxic frame and threedimensional stereotaxic coordinates are selected for placing the needleinto the desired location of the brain or spinal cord.

The donor cells may also be introduced into the putamen, nucleusbasalis, hippocampus cortex, striatum or caudate regions of the brain,as well as the spinal cord.

Preferably, for passaged donor cells, cells are passaged fromapproximately 2 to approximately 20 passages.

For grafting, the cell suspension is drawn up into the syringe andadministered to anesthetized graft recipients. Multiple injections maybe made using this procedure. The age of the donor tissue, i.e. thedevelopmental stage may affect, the success of cell survival aftergrafting.

The cellular suspension procedure thus permits grafting of geneticallymodified donor cells to any predetermined site in the brain or spinalcord, is relatively non-traumatic, allows multiple graftingsimultaneously in several different sites or the same site using thesame cell suspension, and permits mixtures of cells from differentanatomical regions. Multiple grafts may consist of a mixture of celltypes, and/or a mixture of transgenes inserted into the cells.

Preferably from approximately 10⁴ to approximately 10⁸ cells areintroduced per graft.

For transplantation into cavities, which may be preferred for spinalcord grafting, tissue is removed from regions close to the externalsurface of the CNS to form a transplantation cavity, for example asdescribed by Stenevi et al., supra, by removing bone overlying the brainand stopping bleeding with a material such a gelfoam. Suction may beused to create the cavity. The graft is then placed in the cavity. Morethan one transplant may be placed in the same cavity using injection ofcells or solid tissue implants.

Grafting of donor cells into a traumatized brain will require differentprocedures, for example, the site of injury must be cleaned and bleedingstopped before attempting to graft. In addition, the donor cells shouldpossess sufficient growth potential to fill any lesion or cavity in thehost brain to prevent isolation of the graft in the pathologicalenvironment of the traumatized brain.

The present invention therefore provides methods for geneticallymodifying donor cells for grafting CNS to treat defective, diseasedand/or injured cells of the CNS.

The methods of the invention also contemplate the use of grafting oftransgenic donor cells in combination with other therapeutic proceduresto treat disease or trauma in the CNS. Thus, genetically modified donorcells of the invention may be co-grafted with other cells, bothgenetically modified and non-genetically modified cells which exertbeneficial effects on cells in the CNS, such as chromaffin cells fromthe adrenal gland, fetal brain tissue cells and placental cells. Thegenetically modified donor cells may thus serve to support the survivaland function of the co-grafted, non-genetically modified cells, forexample fibroblasts modified to produce nerve growth factor (NGF) invivo as described in the Examples, infra.

Moreover, the genetically modified donor cells of the invention may beco-administered with therapeutic agents useful in treating defects,trauma or diseases of the CNS, such as growth factors, e.g. nerve growthfactor; gangliosides; antibiotics, neurotransmitters, neurohormones,toxins, neurite promoting molecules; and antimetabolites and precursorsof these molecules such as the precursor of dopamine, L-dopa.

The methods of the invention are exemplified by preferred embodiments inwhich donor cells containing vectors carrying a therapeutic transgeneare grafted intracerebrally into a subject. In a first preferredembodiment, the established HPRT-deficient rat fibroblast line 208F,primary rat fibroblasts, and day-1 postnatal primary rat astrocytes wereused to demonstrate that cultured cells genetically modified usingretroviral vectors can survive when implanted in the mammalian brain andcan continue to express transgene products.

In a second preferred embodiment fibroblasts were genetically modifiedto secrete NGF by infection with a retroviral vector, and the modifiedfibroblasts were then implanted into the brains of rats with surgicallesions of the fimbria fornix region. The grafted cells survived andproduced sufficient NGF to prevent the degeneration of cholinergicneurons that would die without treatment. In addition, the protectedcholinergic cells sprouted axons that projected in the direction of thecellular source of NGF.

In a third preferred embodiment fibroblasts were genetically modified toexpress and secrete L-dopa by infection with a retroviral vector, andthe modified fibroblasts were grafted into the caudate of rats modelingParkinson's disease as a result of unilateral dopamine depletion. Thecells survived and produced sufficient L-dopa to decrease the rotationalmovement caused by dopamine depletion.

In a fourth preferred embodiment primary skin fibroblasts isolated fromskin biopsies and maintained in culture were employed as autologouscells for intracerebral grafting into the adult rat striatum. Thefibroblasts ceased to proliferate once they reached confluency and werecontact inhibited in vitro. The fibroblasts were able to survive for atleast eight weeks following intracerebral implantation and continued tosynthesize collagen and fibronectin in vivo. The grafts also maintaineda constant volume between three and eight weeks, indicating that theprimary skin fibroblasts did not tumor or die. Dynamic host-to-graftinteractions, including phagocytic migration, astrocytic hypertrophy andinfiltration into the grafts, and angiogenesis, were observed indicatingthe structural integration of grafts of primary skin fibroblasts in theadult rat CNS.

In a fifth preferred embodiment primary skin fibroblasts obtained from askin biopsy from an inbred strain of rats were used as donor cells forgenetic modification and grafting. When grafted into the brain of ratsof the same genetic strain as the donor rats, the fibroblasts containinga transgene for either tyrosine hydroxylase (TH) or β-galactosidasesurvived for 10 weeks and continued to express the transgene. The THsynthesized by the implanted fibroblasts appeared to convert tyrosine toL-dopa, as observed in vitro, and to affect the host brain as assessedthrough a behavioral measurement. Supplying L-dopa locally to thestriatum was shown to be sufficient to partially compensate for the lossof striatal dopaminergic input.

In a sixth preferred embodiment, the ability of aged human fibroblaststo serve as donor cells for human NGF was demonstrated.

In a seventh preferred embodiment rat fibroblasts were geneticallymodified to express and secrete choline acetyltransferase (dChAT). Itwas shown that intra- and extracellular levels of ACh could be increasedby adding exogenous choline chloride. In addition, serum starvation orconfluence-induced quiescence caused an 80% decrease in recombinant ChATactivity (as compared to actively growing cells). ACh release was alsorepressed in quiescent fibroblast cultures. Exogenous choline mitigatedthe decrease in ACh secretion. These results indicated that fibroblastscan be genetically modified to produce ACh and that ACh release can beregulated, for example increased, by introducing choline into theculture medium.

In an eighth preferred embodiment selected promoters including collagenpromoters were demonstrated to increase expression of the transgenechloramphenicol acetyltransferase (CAT) in quiescent primaryfibroblasts.

In a ninth preferred embodiment cytokines and an anti-inflammatoryagent, dexamethasone, were evaluated for their effect on LTR promoteractivity to enhance expression of the CAT and dChAT genes in primaryskin fibroblasts. In addition, the collagen promoter α2(I) with thecollagen enhancer (Coil(E)) was used to evaluate the effects ofcytokines on the expression of CAT in quiescent fibroblasts.

In an tenth preferred embodiment primary skin fibroblasts were modifiedto express and secrete NGF and were grafted into the striatum of adultfemale rats. At one and three weeks following striatal implantations,NGF receptor-immunoreactive axons surrounded the grafts of NGF-producingfibroblasts. At three and eight weeks, NGF receptor-positive profileswere also found within the grafts. Control grafts of normal primaryfibroblasts lacked immunoreactive axons. Ultrastructural examinationshowed that unmyelinated axons within the extracellular matrix of thegrafts were enveloped within processes of reactive astrocytes and adistinct basal lamina surrounded the axo-glial bundles. Processes ofreactive astrocytes were evident in both control and grafts containinggenetically modified primary fibroblasts. This embodiment demonstratesan in vivo model showing that the release of NGF from the grafts induceddirectional growth of NGF receptor-positive axons and that maturereactive astrocytes provide a permissive substrate upon which axonsmigrated. In this embodiment, genetically modified fibroblasts were alsoplaced in a collagen matrix graft to assess the regenerative capacity ofthe adult rat medial septum. The grafts produced NGF and promoted theregeneration of septal axons; the grafts possessed large numbers ofunmyelinated axons compared to control fibroblast grafts. Theregenerating septal axons provided a reinnervation to the deafferentedhippocampus; the topographical and synaptic organization of the septalinputs within the hippocampal dentate gyrus was similar to that of thenormal cholinergic innervation arising from septal nuclei.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting the scope of this invention in anymanner.

EXAMPLE I Intracerebral Grafting Of Genetically Modified CellsExpressing HPRT Transgene To The Brain

Infection of Cells

Donor hypoxanthine quanine phosphoribosyl transferase (HPRT)-deficient208F rat fibroblast cells (jolly et al., Proc. Natl. Acad. Sci, USA 80:477-481 (1983)) were infected with the prototype HPRT vector pLSΔPΔLMexpressing HPRT cDNA (Miller et al., Science 225: 630-632 (1984); Yee etal., Gene 53: 97-104 (1987)) cDNA or with the neo^(R) -luciferase vectorpLNHL2 (Eglitis et al., Science 230: 1395-1398 (1985); Wolff et al.,Proc. Natl. Acad. Sci. (USA) expressing both the Tn5 transposonneomycin-resistance gene (neo^(R)) and the firefly luciferase cDNA (deWet et al., Molec. Cell. Biol. 7: 725-737 (1987)) (FIG. 4). The pLSΔPΔLMvector was derived from vector pLPL2 (Miller et al., Molec. Cell. Biol.6: 2895-2902 (1986)) and contains human HPRT cDNA encoding a proteinwith a novel C-terminal hexapeptide added by in vitro mutagenesis of thetranslational termination codon (Yee et al., Gene 53: 97-104 (1987)).Vector pLSΔPΔLM was constructed as follows: Vector pLpLM (Yee et al.,Gene 53: 97-104 (1987)) was digested with Xhol and BamHl to yield a 1.3Kb fragment which was then ligated into plasmid pLpL2 (Miller, supra)which had also been restricted using Xhol and BamHl. The resultingvector was pLS P LM.

The vector pLNHL2 contained the cDNA encoding the firefly luciferase(LUX) and the Tn5 neomycin-resistance gene (neo^(R)) and the promoterand enhancer of the human cytomegalovirus immediate early gene (HCMV).The 5' and 3' LTRs were derived from cloned murine leukemia virus (MLV)as described by Mason et al., in Science 234: 1372-1378 (1986). VectorpLNHL2 was constructed as follows: Plasmid pLNHPL2 (also known asPNHP-1, Yee et al., Proc. Natl. Acad. Sci. USA 84: 5197-5209 (1987)) wasrestricted with BamHI to remove the HPRT DNA sequence. The ends wererepaired using Klenow polymerase. Plasmid pSV2A (deWet et al., Molec.Cell. Biol., supra, and supplied by) (Dr. Subramani, University ofCalifornia, San Diego, Calif.) was restricted with HindIII and Sspl toisolate the luciferase fragment. The ends were repaired as above. TheBamHl restricted pLNHL2 and HindIII-Sspl restricted pSV2A were ligatedtogether forming vector pLNHL2 (FIG. 5).

The cells were grown in selective medium containing hypoxanthine,aminopterin and thymidine (HAT) for cells expressing HPRT and with theneomycin analog G418 for cells expressing neo^(R), respectively, toensure that only infected cells were used.

Primary fibroblasts and astrocytes were infected with the neo^(R)-luciferase vector only. HAT-resistant and G418-resistant cells wereharvested following incubation overnight with serum-free medium ormedium containing rat serum, to reduce the likelihood of immunologicalresponse in the rat brain.

Grafting

The cells were resuspended in a balanced glucose-saline solution andinjected stereotaxically into several regions of the rat brain using asterile syringe. Between 10,000 and 100,000 cells per microliter wereinjected at a rate of 1 μl/min for a total volume of 3-5 μ. After 1 weekto 3 months the animals were killed and areas containing the implantedcells were identified, excised, and examined histologically andbiochemically.

Histological Analyses

To evaluate the grafted cells histologically, the rats were perfusedtranscardially and their brains were sectioned and stained with Nisslstain and cresyl violet for general morphological characterization andwith immunocytochemical methods to establish the presence of thespecific cell antigenic markers fibronectin for the fibroblasts andglial fibrillary acidic protein (GFAP) for glial cells. Briefly, thesections were rinsed in Tris-buffered saline (TBS) solution (pH 7.4)containing 0.25% Triton-X. The sections were incubated for 24 hrs at 4°C. with rabbit polyclonal antibodies to fibronectin (1:2000 dilution;Baralle, University of Oxford, England) and GFAP (Gage et al., Exp.Neurol. 102: 2-13 (1989); available from Dakopatts, Glostryp, Denmark)diluted 1:1000 in TBS containing 0.25% Triton-X and 3% goat serum orwith the monoclonal antibody, mouse IgG2a, against a membranepolypeptide of rat macrophages, granulocytes and dendritic cells (MRCOX-42, Serotec) diluted 1:100 in TBS containing 0.25% Triton-X and 1%horse serum. After thorough rinsing, the sections were incubated for 1hr with biotinylated goat anti-rabbit IgG (Vectastain) diluted 1:200 in0.1M TBS containing 0.25% Triton-X and 15 horse serum, followed byseveral rinses in TBS containing 0.25% Triton-X and 1% goat serum or 1%horse serum. The sections were then incubated for 1 hr at roomtemperature with a complex of avidin and biotinylated horseradishperoxidase (Vectastain, ABC kit, Vector Labs, Burlingame, Calif.)diluted 1:100 in 0.1M TBS containing 0.25% Triton-X and 1% goat serum or1% horse serum, followed by thorough rinses. The peroxidase wasvisualized by reacting with 0.05%, 3,3-diaminobenzidinetetrahydrochloride (DAB) (Sigma Chemical Co., St. Louis, Mo.) and 0.05%NiCl₂ and 0.01% H₂ O₂ in TBS for 15 min at room temperature.

Primary rat fibroblasts grafted to the neostriatum of the rat sevenweeks earlier are illustrated in FIG. 6. Serial 40 μm-thick sectionswere stained with antifibronectin (FIG. 6a, FIG. 6d), cresyl violet(Disbrey et al., Histological Laboratory Methods, E. & S. Livingstone,Edinburgh and London (1970)), (FIG. 6b, FIG. 6e) and anti-GFAP (FIG. 6c,FIG. 6f). The surviving cells appeared to be intact and to have clumpedor aggregated around the area of the injection. The cells displayed anintense staining for fibronectin at the core of the graft, with a clearGFAP-staining derived from reactive gliosis at the edges of the grafts,similar to what one sees with the cannula tract alone. However, littleGFAP-staining was observed in the graft itself. With cresyl violet,small, round, darkly stained cells were observed in the region of thegraft which could either be microglia or lymphocytes that hadinfiltrated the area in response to injury. Macrophages could also bedetected in many of the grafts. Many of the fibroblasts could beidentified by cresyl violet staining by their long thin shape and by thepink pleated sheets of collagenous material surrounding them. Theappearance of 208F fibroblasts was similar to the primary fibroblasts(not shown). Astrocyte grafts also had a similar appearance, except theywere not fibronectin-positive, and stained for GFAP through the centerof the grafts. For all three cell types, no differences were observedbetween retrovirus-infected cells and control cells. An importantfeature of these cell suspension grafts is that most of the cellsremained aggregated near the site of injection and did not appear, underthese circumstances, to migrate very far from the injection site intothe host brain. This apparent lack of migration could certainly bedifferent for other donor cell types and graft sites, and therefore thearea of the brain into which the cells are to be implanted, the natureof the donor cells, and the phenotype of the target cells for thetransgene may be important factors for the selection of donor cells.

Characterization of Implanted Cells

Implanted cells were dissected out and prepared for reculturing and forbiochemical and molecular characterization by dissociating the cellswith trypsin. For the detection of the human HPRT activity, cellextracts were prepared from the bulk of each sample as previouslydescribed and examined by a polyacrylamide gel isoelectric focusing HPRTassay (Jolly et al., Proc. Natl. Acad. Sci. USA) 80: 477-481 (1983);Miller et al., Proc. Natl. Acad. Sci (USA) 80: 4709-4713 (1983); Williset al., J. Biol. Chem. 259: 7842-7849 (1984); Miller et al., Science225: 630-632 (1984); Gruber et al., Science 230: 1057-1061 (1985), andYee et al., Gene 53: 97-104 (1987)). The remainder of each sample wasplaced into culture. The results of an HPRT gel assay of rat 208F cellsHAT resistance after infection with the HPRT vector implanted into oneside of the rat basal ganglia 3 and 7 weeks after transplantation andprior to analysis are shown in FIG. 7.

The presence of human HPRT enzyme activity demonstrates that theinfected, genetically modified rat 208F cells grafted into the brainsurvived and continued to express the HPRT transgene at easilydetectable levels for at least 7 weeks. Furthermore, the implanted cellscould be successfully recultured, producing cells morphologicallyidentical to the starting cultures. Infection of these cells with helpervirus resulted in the production of HPRT virus, confirming the identityof the cells and indicating that the provirus remained intact. Studieswith the neo^(R) -luciferase vector confirm the survival and expressionof luciferase-infected cells.

EXAMPLE II Grafting of Genetically Modified Cells Expressing NGF to theDamaged Brain

The above example demonstrated that cultured cells genetically modifiedusing retroviral vectors can survive when implanted into the mammalianbrain and can continue to express transgene product. The present examplewas conducted to determine whether sufficient transgene product can bemade by genetically modified cells in vivo to complement or repair anabsent or previously damaged brain function.

Construction of NGF Vector pLN.8RNL

A retrovital vector, similar to one described previously (Wolf, et al.,Mol. Biol. Med. 5: 43-59 (1988)), was constructed from Moloney murineleukemia virus (MoMuLV) (Varmus et al., RNA Tumor Viruses; Weiss et al.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p. 233 (1982)). ThepLN.8RNL vector contains the 777 base pair Hgal-Pstl fragment of mouseNGF cDNA (Scott et al., Nature 302; 538 (1983); Ullrich et al., Nature303: 821 (1983)), under control of the viral 5' LTR. This insertcorresponds to the shorter NGF transcript that predominates in mousetissue receiving sympathetic innervation (Edwards et al., Nature 319:784 (1986)) and is believed to encode the precursor to NGF that issecreted constitutively. The vector also included a dominant selectablemarker encoding the neomycin-resistance function of transposon Tn5(Southern et al., J. Mol. Appl. Genet. 1: 327 (1982)), under control ofan internal Rous sarcoma virus promoter.

Plasmid pSPN15' (Wolf et al., Mol. Biol. Med. 5: 43-59 (1988), suppliedby Dr. Breakefield, Harvard Medical School, Boston, Mass.) was digestedwith restriction enzymes Pstl and Hgal using established methods(Maniatis et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,(1982)) and the 777 base pair (bp) DNA fragment containing the NGFsequences was isolated by standard purification methods (Maniatis, etal., supra). The 777 bp fragment was then blunt-end ligated into plasmidpLMTPL as described by Yee et al., in Proc. Natl. Acad. Sci. (USA) 84:5197-5201 (1987), incorporated by reference herein. Plasmid pLMTPL wasdigested with Hind III to remove the metallothionein promoter and mostof the HPRT cDNA, and the overhanging 5' ends were repaired using Klenowpolymerase as described by Maniatis et al., supra. The overhanging endsof the 777 bp fragment isolated as above were similarly repaired. The777 bp fragment was then blunt end ligated into the digested plasmidpLMTPL. The resulting plasmid was called PLN.8L and was transfected intoE. coli strain DHl, grown and purified by established methods forplasmid purification including cesium chloride centrifugation (Maniatiset al., supra) . The purified plasmid pLN.8L was then digested usingrestriction enzymes BamHI and Clal and the resulting 6.1 kilobasefragment was ligated to a 2.1 kilobase BamHl-Clal fragment isolated fromplasmid pLLRNL. Plasmid pLLRNL was derived from plasmid JD204 describedby de Wet et al., in Mol. Cell. Biol. 7: 725-737 (1987) as follows: A1717 bp HindIII-Sspl fragment from the firefly luciferase gene derivedfrom plasmid JD204 and a 1321 bp HindIII-Smal fragment of the plasmidpSV2Neo^(R) described by Southern and Berg in Mol. Appl. Genet. 1:327-341 (1987) were ligated with a fragment containing a mutated RSVpromoter in a 300 bp BamHI-HindIII fragment from plasmid pUCRH. PlasmidpLLRNL is depicted in FIG. 8.

Plasmid pUCRH was produced as follows. Plasmid pRSV neo^(R) wasrestricted using HindIII and the linearized plasmid was ligated with theremaining fragment from plasmid pPR1 (FIG. 9) (supplied by Dr.Friedmann, University of California, San Diego, Calif.) obtained byrestriction using HindIII to remove HPRT sequences. Plasmid pPR1 wasobtained from plasmid p4aA8 (Jolly et al., Proc. Natl. Acad. Sci. 80:477-481 (1983)) using Pst and Rsa. The resulting plasmid was called pRHNand was then restricted with PvuII and religated to form plasmids PN(+)and PAC(-). PN(+) was then restricted using BamHl. The resultinglinearized plasmid was ligated with a fragment obtained from plasmidpSVori restricted with BamHI. Plasmid pSVori was obtained by restrictingplasmid p4aA8 with Sall and Pstl, and subcloning the resulting fragmentinto plasmid pUC18 (Bethesda Research Laboratories, Gaithersburg, Md.)that had been restricted with Sall and Pstl. The resulting plasmid wastermed pSVori.

The plasmid pRH+S+that resulted from ligation of the BamHl restrictedplasmid PN(+) and the BamHl restricted plasmid pSVori was thenrestricted with MstII and the overhanging 5' ends were repaired usingKlenow polymerase as described above. This fragment was ligated with aM13mp18 (Bethesda Research Laboratories) linearized with Smal andphosphatased with calf intestinal alkaline phosphatase (BoehringerMannheim, Mannheim, W. Germany). The resulting plasmid was called pmpRHand contained the HPRT cDNA expressed from the RSV promoter.

Plasmid pmpRH was subject to site-directed mutagenesis as described byKunkel et al., Proc. Natl. Acad. Sci. USA 82: 488-492 (1985),incorporated by reference herein in order to alter the polyadenylationsignal AATAAA to AGCAAA. After mutagenesis the resulting plasmid wasrestricted using HindIII and the resulting fragment was ligated to aHindIII fragment from restriction of plasmid pUC19 (Bethesda ResearchLaboratories) to produce plasmid pUCRSV. Plasmid pUCRSV was restrictedusing BamHl and Pstl to produce a fragment containing the RSV promoter.This fragment was ligated to a Pstl-Sstl fragment containing the geneencoding HPRT obtained by restriction of the plasmid pLS P LM asdescribed in Example I, supra and to a BamHI-Sstl fragment obtained fromplasmid pUC19, forming plasmid pUCRH (FIG. 10). The product of ligationbetween the BamH1-Hindlll fragment from pUCRH, the 1717 bp HindIII-Ssplfragment from pJD204 and the 1321 bp HindIII-Smal fragment frompSV2Neo^(R) was transfected and purified by established methods asdescribed above and termed plasmid pLLRNL (FIG. 8).

After transfection and purification, the plasmid resulting from ligationof ,the 6.1 BamHl-Clal kilobase fragment from plasmid pLN.8L and the 2.1kilobase BamHl-Clal fragment from plasmid pLLRNL was termed pLN.8RNL.(FIGS. 11 and 12).

Preparation of Transmissible Retrovirus

Transmissible retrovirus was produced by transfecting pLN.8RNL intoPA317 amphotropic producer cells (Miller et al., Mol. Cell. Biol. 6:2895 (1986)), supplied by Dr. Miller, Fred Hutchinson Cancer ResearchCenter, Seattle, Wash.), by the calcium phosphate co-precipitationmethod (Graham et al., Virology 52: 456 (1973)), and using medium fromthese cells to infect Ψ-2 ecotropic producer cells (Mann et al., Cell33: 153 (1983)) in the presence of 4 μg/ml Polybrene (Sigma Chemical,St. Louis, Mo.). Virus from the Ψ2 clone producing the highest titer,4×10³ colony forming units/ml, was used to infect the established ratfibroblast cell line 208F (Quade, Virology 98: 461 (1979) as describedby Miyanohara et al., in Proc. Natl. Acad. Sci. USA (1988)).

Assay for NGF Production and Secretion

Individual neomycin-resistant 208F colonies, selected in mediumcontaining the neomycin analog G418, were expanded and tested for NGFproduction and secretion by a two site (ELISA) enzyme immunoassay(Korsching et al., Proc. Natl. Acad. Sci. (USA) 80: 3513-3516 (1983)),using commercially available reagents according to the manufacturer'sprotocol (Boehringer Mannheim). The clone producing the highest levelsof NGF contained 1.7 ng NGF/mg total cellular protein and secreted NGFinto the medium at a rate of 50 pg/hr 10⁵ cells. The NGF secreted bythis clone was biologically active, as determined by its ability toinduce neurite outgrowth from PCl2 rat pheochromocytoma cells (Greene,et al., Proc. Natl. Acad. Sci. USA 73: 2424 (1976)). Uninfected 208Fcells, in contrast, did not produce detectable levels of NGF in eitherassay.

Fimbria Fornix Transection

Fimbria fornix transection (axotomy) was performed as described by Gageet al., Brain Res. 268: 27-37 (1983) and in Neuroscience 19(1) 241-255(1986), both of which are incorporated by reference herein. Briefly,adult female Sprague-Dawley rats (Bantin and Kingman, San Francisco,Calif.) weighing between 200 g and 225 g at the beginning of theexperiment were used. The animals were anesthetized with intraperitonealinjections of a ketamine-xylazine mixture (10 μg/kg Ketalar, Parke-DavisAnn Arbor, Mich., and 5 μg/kg Rompun, Hoechst, Frankfurt, W. Germany).Unilateral aspirative lesions were made by suction through the cingulatecortex, completely transecting the fimbria fornix unilaterally, andremoving the dorsal tip of the hippocampus as well as the overlyingcingulate cortex to inflict a partial denervation on the hippocampustarget, as described in Gage et al., Brain Res. 268: 27-37 (1983). Allanimals in each of the experimental groups received the same completeunilateral aspirative lesion. Fimbria fornix lesions as described abovewere made in 16 rats; 8 rats received grafts of infected cells while theremaining 8 received uninfected control cells.

Sections stained for fibronectin, a fibroblast specific marker, revealedrobust graft survival that was comparable in both groups (FIG. 13a, FIG.13b). Sections stained for choline acetyltransferase (CHAT) to evaluatethe survival of cholinergic cells bodies indicated a greater number ofremaining neurons on the lesioned side of the medial septum in animalsthat had received grafts of infected cells than in animals that hadreceived control grafts (FIG. 13c-FIG. 13f).

Retrovirus-infected (NGF secreting) and control 208F cells were removedfrom confluent plates with Dulbecco's phosphate buffered saline (PBS)containing 0.05% trypsin and 1 mM EDTA and taken up by trituration withPBS supplemented with 1 mg/ml glucose, 0.1 mg/ml each MgCl₂ and CaCl₂(complete PBS) and 5% rat serum to inactivate the trypsin. Cells werepelleted by centrifugation at 1000 X g for 4 min. at 4° C., washed twicewith complete PBS, and resuspended in complete PBS at 105 cells/μl. Fourμl of suspended cells were injected free-hand using a Hamilton syringeinto the cavity and lateral ventricle ipsilateral to the cavity in theanimals. A piece of Gelfoam was gently placed on the surface of thecavity and the animals were sutured.

Immunohistochemistry

At 2 weeks following surgery the rats were perfused and their brainswere removed, fixed overnight and placed in phosphate-buffered 30%sucrose for 24 hr at 4° C. Sections 40 μm thick were cut on a freezingsliding microtome and stored in cryoprotectant (phosphate-bufferedglycerol and ethylene glycol) at -20° C. Every fifth section waslabelled immunohistochemically by standard procedures using polyclonalantibodies to fibronectin to evaluate fibroblast survival. Polyclonalantibodies to choline acetyltransferase (anti-ChAT antiserum) were alsogenerated to evaluate the survival of cholinergic cell bodies asdescribed by Gage et al. in J. of Comparative Neurol. 269: 147-155(1988), incorporated by reference herein. Tissue sections were processedfor immunohistology according to a modification of the avidin-biotinlabeling procedure of Hsu et al., 29: 1349-1353(1981), incorporated byreference. This procedure consists of the following steps: 1) overnightincubation with antibody to ChAT or with control antibody (i.e.preimmune serum or absorbed antiserum). The ChAT antibody was diluted1:1,500 with 0.1 M Tris-saline containing 1% goat serum and 0.25% TritonX-100; 2) incubation for 1 hr with biotinylated goat anti-rabbit IgG(Vector Laboratories, Burlingame, Calif.) diluted 1:200 with Tris-salinecontaining 1% goat serum: 3) 1 hr incubation with ABC complex (VectorLaboratories) diluted 1:100 with Tris-saline containing 1% goat serum;4) treatment for 15 min with 0.05% 3,3-diaminobenzidine (DAB), 0.01%hydrogen peroxide and 0.04% nickel chloride in 0.1M Tris buffer.Immunolabeled tissue sections were mounted onto glass slides, air driedand covered with Permount and glass coverslips. Two sections stained forChAT through the septum, 200 μm apart were used to evaluate the extentof cholinergic cell survival. All the ChAT-positive cells in theipsilateral septum and in the contralateral septum were countedseparately and sized for planar area using an Olympus Que-2 imageanalysis system. Tissues were also stained for acetylcholinesterase(ACHE) as described by Hedreen et al., J. Histochem. Cytochem. 33:134-140 (1985), incorporated by reference herein, to evaluate thecompleteness of the fimbria fornix transection.

Neuronal survival was quantitated (FIG. 14) and, when expressed as apercentage of the remaining cholinergic cells in the septum ipsilateralto the lesion relative to the intact contralateral septum, was shown tobe 92% in animals grafted with NGF-secreting cells but only 49% inanimals grafted with control cells. The results from the control groupare comparable to previous observations in lesioned animals that hadreceived no grafts (Gage et al., Neuroscience 19: 241 (1986); Hefti, J.Neurosci. 8: 2155 (1986); Williams et al., Proc. Natl. Acad. Sci. USA83: 9231 (1986); Kromer, Science 235: 214 (1987); Gage et al., J. Comp.Neurol. 369: 147 (1988)).

In addition to the significant increase in the percentage ofChAT-positive cells in the NGF group, these animals also showed anincrease in AChE-positive fiber and cell staining (FIG. 15). Moststriking was the observation of a robust sprouting response in thedorsal lateral quadrant of the septum, with the most intense stainingabutting the cavity containing the graft. This intense increase in AChEstaining was not observed in the group receiving control grafts (FIG.15).

The above results demonstrate the feasibility of continued transgeneexpression by cells grafted to the CNS and also present the firstdemonstration of a phenotypic correction in whole animals brought aboutby grafted, genetically modified cells.

EXAMPLE III Grafting of Genetically Modified Fibroblasts ExpressingL-dopa Into The CNS of A Rat Model of Parkinson's Disease

This example was undertaken to demonstrate that the methods of thepresent invention for genetic modification of donor cells and graftingof the cells into the CNS can significantly ameliorate the signs ofdisease in an animal model, such as a rat model of Parkinson's disease.

The strategy for enabling fibroblasts to produce L-dopa used in thisexample is based upon the ability of the enzyme tyrosine hydroxylase(TH) to catalyze the conversion of tyrosine to L-dopa; the rate-limitingstep in catecholamine synthesis. Tetrahydrobiopterine (H₄ -B), theco-factor for TH is required for TH enzymic activity. Since the braincontains significant levels of biopterin, and fibroblasts can reducebiopterin to H₂ -biopterin, TH should be active in fibroblasts situatedwithin the brain.

Construction of Retroviral Vector pLThRNL

The vector pLThRNL, a Moloney leukemia virus (MoMLV) derived retroviralvector, was constructed expressing the rat cDNA for tyrosine hydroxylase(TH) from the 5' LTR sequence and contained a neomycin-resistant genetranscribed from an internal RSV promoter (FIG. 16). Fragments fromthree plasmids: pLRbL, pTH54 and pLHRNL were ligated together to formpLThRNL. Plasmid pLRbL was obtained by digesting plasmid pLMTPL(obtained as described above in Example II) with the enzymes HindIII andHpal, and removing the fragment containing the HPRT gene. The remainingplasmid DNA was ligated with the 3.5 kb fragment obtained afterrestriction of plasmid pGEM1-4.5Rb old (pGEM1-4.5Rb old was constructedby inserting DNA encoding the retinoblastoma gene (Rb) into plasmidpGEMl, available from Promega, Madison, Wis., and was supplied by Dr.Lee, University of California, San Diego, Calif.) using HindIII andSca2. The resulting plasmid was named pLRbL.

A 1688 bp fragment containing rat TH cDNA was obtained from the plasmidpTH54 (O'Malley, J. Neurosci. Res. 60: 3-10 (1986), supplied by Dr.O'Malley, Washington University, St. Louis, Mo.) by digestion withBamIII and Sphl.

The fragments from plasmid pTH54 and plasmid pLRbL were ligated with aClal and Smal fragment obtained from plasmid pL2RNL (described above inExample I) to obtain the vector pLThRNL containing the retroviralprovirus for transfection into producer cells to produce virus carryingthe gene encoding the enzyme tyrosine hydroxylase. The derivation of andcircular restriction map for pLThRNL is shown in FIG. 17.

Helper-free retrovirus was produced and retroviral infections were doneas described in Example II, supra Plasmid DNA containing the LThRNLprovirus was CaPO₄ transfected as described by Wigler et al., Cell 11:223-232 (1977), incorporated by reference herein, into the amphotropicPA317 helper line supplied by Dr. Miller, Fred Hutchinson CancerResearch Center, Seattle, Wash. Two days post-transfection, media fromthese cells were filtered and used to infect the ecotropic Ψ2 helperline (Miller et al., Mol. Cell. Biol. 6: 2895-2902 (1986), supplied byDr. Miller). A G418-resistant Ψ2 clone (Ψ2/TH) that contained thehighest level of TN activity and produced the highest titre of virus(5×10⁵ /ml) was selected. Immortalized, rat fibroblasts (208F) (Quade,Virology 98: 461-465 (1979)) were infected at a multiplicity ofinfection (MOI) of less than 10⁻⁴ with LThRNL virus produced by theΨ2/TH producer cells. G418 resistant clones were established for furtherstudy. All retroviral infections were done in the presence of 4 μg ofPolybrene (Sigma) per ml. Cells were selected for expression of theneomycin-resistance gene by growth in 400 μg/ml of G-418.

Assay of Tyrosine Hydroxylase Activity

Confluent 10 cm plates of cells were washed two times with Dulbecco'sphosphate buffered saline (PBS) not containing calcium or magnesiumchloride and the cells were scraped off the plates. The cells werehomogenized in 0.15 ml of ice cold 50 mM Tris/50 mM sodiumpyrophosphate/0.2% Triton X-100, adjusted to pH 8.4 with acetic acid,and were centrifuged at 32,000 X g for 15 min at 2°-4° C. Thesupernatant fraction was used for both TH and protein measurements. THactivity was measured with a decarboxylase-coupled assay essentially asdescribed previously (Iuvone et al., J. Neurochem, 43: 1359-1368(1986)), but with. ¹⁴ C-labelled 20 μM tyrosine, 1 mM6-methyl-5,6,7,8-tetrahydropterin (6MPH4) (Calbiochem, La Jolla,Calif.), and potassium phosphate buffer (pH 6). Protein was determinedby the method of Lowry et al. (J. Biol. Chem. 193: 265 (1951)) usingbovine serum albumin as standard.

Assays of Catecholamines and their Metabolites

Cultured cells were scraped off plates as in the assay of tyrosinehydroxylase activity except ascorbic acid (final concentration of 50 nM)was added to the cell pellets prior to freezing. Cells were grown in DMEplus 10% fetal calf serum. Some cultures were supplemented with 0.1mM6MPH4, 16 hours prior to harvesting. Cells were homogenized in 250 μl ofice cold 0.1 N HCl/0.1% sodium metabisulfite/0.2% Na₂ EDTA containing 5ng/ml of 3,4-dihydroxybenzylamine (DHBA) as internal standard. Dopamine,DOPA, 3,4-dihydroxyphenylacetic acid (DOPAC), DHBA and3-methoxy-4-hydroxyphenylglycol (MHPG), were extracted from thesupernatant fraction by alumina absorption (Anton et al., J. Pharmacol.Exp. Ther. 138: 360-375 (1962)) and eluted with 150 μl of 0.1NH₂ PO₄.They were analyzed by HPLC with electrochemical detection as describedby Iovone et al., Brain Res. 418: 314-324 (1987), with the mobile phasemodified to contain a higher concentration of sodium octylsulfate (0.45mM) and lower pH (2.8). Homovanillic acid (HVA) was. analyzed by HPLC.The concentration of catecholamines and metabolites in the media wasalso determined using a different method, HPLC-EC. The aluminaextraction procedure described above was omitted and the media wasadjusted to 0.1 perchloric acid/0.01M EDTA was centrifuged 10,000g x 10min to remove precipitated material and used directly for HPLC-EC. Inthis system, the whole phase consisted of 0.137% SDS in 0.1M phosphatebuffer, pH 3.2 (Buffer A) or 40% methanol in 0.1M phosphate buffer, pH3.35 (Buffer B). Compounds in sample were eluted for 12 minutes in 100%Buffer A, followed by a gradient increasing linearly over 30 minutes to100% Buffer B. The eluant was then passed through a series of 16coulometric electrodes set at 60 mV increments.

Rat Model of Parkinson's

Female Sprague-Dawley rats received a unilateral injection of 12 μg in 2μl saline-ascorbate 6-hydroxydopamine (6-OHDA) into the medial forebrainbundle (coordinates: AP=-4.4; ML=i.1; DV=7.5). Completeness of thelesion produced was assessed 10 to 20 days postinjection by eitherapomorphine (0.1 mg/kg, subcutaneously (s.c.)) or amphetamine (5 mg/kg,s.c.) induced rotational behavior (Ungerstedt and Arbuthnott, Brain Res.24: 485-493 (1970)). Prior to transplantation, each animal was tested atleast twice on separate days to establish the baseline rotationalresponse to apomorphine or amphetamine for each animal. Animals turningat a rate of more than 7 turns per minute (Schmidt et al., J. Neurochem.38: 737-748 (1982)) were included in the study (at least 7 contralateralrotations/min following apomorphine administration and at least 7ipsilateral rotations/min towards the side of the lesion followingamphetamine administration; 19 apomorphine tested, 14 amphetaminetested). The average percent change in the number of rotations frombaseline to post-transplantation was compared in the 4 experimentalgroups of animals.

Grafting of Fibroblasts

Confluent 10 cm plates of cultured TH-infected or noninfectedfibroblasts were loosened from the plates in PBS containing 0.05%trypsin and pipetted up in PBS supplemented with 1 mg/ml glucose, 0.1mg/ml MgCl₂ and 0.1 mg/ml CaCl₂ (complete PBS) plus 5% rat serum toinactivate the trypsin. The cells were washed twice with complete PBSusing centrifugation at 1000 X g and were resuspended in complete PBS ata density of 80,000 cells per μl. Since graft placement has been shownto be crucial for recovery from rotational asymmetry (Herrera et al.,Brain Res. 297: 53-61 (1984); Dunnett et al., Scand. Suppl. 522: 29-37(1983)), suspended cells were injected stereotaxically into 2 to 3separate locations within the rostral (coordinates: AP=i.4; ML=2.0;DV=3.5-5.5 to AP=2.5; ML=i.5; DV=3.5/4.5) and caudal areas (AP=0.4;ML=3.0; DV=3.5/4.5) of the denervated caudate. A total of 4 μl weredelivered in two equal deposits over a 1 to 2 mm area at each site.Control lesioned animals received injections of noninfected fibroblasts.

Post-Grafting Behavioral Testing

Grafted rats were tested for rotational asymmetry 1 and 2 weeksfollowing fibroblast grafting.

Histological Methods

Following the final behavioral test, rats were deeply anesthetized andperfused with 10% formalin. Brains were postfixed overnight, placed in30% sucrose for 48 hrs and then sectioned (40 μm) on a freezingmicrotome. Alternate sections were stained for cresyl violet,fibronectin (FB) or TH using a polyclonal anti-tyrosine hydroxylaseantibody (Eugenetech, N.J.). Briefly, the sections were rinsed inTris-buffered saline (TBS) solution (pH 7.4) containing 0.25% Triton-X.The sections were incubated for 24 hrs at 4° C. with rabbit polyclonalantibodies to tyrosine hydroxylase diluted 1:600 or polyclonalanti-fibronectin diluted 1:2000 in TBS containing 0.25% Triton-X and 3%goat serum. After thorough rinsing, the sections were incubated for 1 hrwith biotinylated goat anti-rabbit IgG (Vectastain) diluted 1:200 in0.1M TBS containing 0.25% Triton-X and 1% goat serum, followed byseveral rinses in TBS containing 0.25% Triton-X and 1% goat serum. Thesections were then incubated for 1 hr at room temperature with a complexof avidin and biotinylated horseradish peroxidase (Vectastain, ABC kit,Vector Labs, Burlingame, Calif.) diluted 1:100 in 0.1M TBS containing0.25% Triton-X and 1% goat serum, followed by thorough rinses. Theperoxidase was visualized by reacting with 0.05%, 3,3-diaminobenzidinetetrahydrochloride (DAB) (Sigma Chemical Co., St. Louis, Mo.) and 0.05%NiCl₂ and 0.01% H₂ O₂ in TBS for 15 min at room temperature. Mountedsections were assessed for size and placement of fibroblast positivegrafts.

Establishment of a Fibroblast Clone Expressing High Levels of TH

Immortalized, rat fibroblasts (208F) were infected with LThRNL virusproduced by the Ψ2/TH producer cells and 12 G418-resistant clones wereestablished. Table 1 shows the TH activity of 3 of these 12 G418resistant clones with the highest TH activity and the TH activity of theΨ2 producer line. The TH activity of the clones with the highestactivity (clones 208F/TH-8 and 208F/TH-11) contained approximately aquarter of the TH activity of rat striatum. The 208F/TH-8 clone thatcontained the highest TH activity, was chosen for further study.

                  TABLE 1                                                         ______________________________________                                        TH Activity of Cell and Tissue Extracts                                       Cell Line      TH Activity*                                                   ______________________________________                                        ψ2/TH      1.7                                                            208F/TN-8      2.9                                                            208F/TH-11     2.6                                                            208F/TH-9      0.4                                                            208F/CONTROL   0.0                                                            Rat Striatum   9.8                                                            ______________________________________                                         *TH activity is expressed in units of pmoles DOPA/min/mg protein         

Fibroblasts Expressing TH Produce and Secrete L-dopa.

Cell extracts from the 208/TH-8 fibroblasts expressing TH and control208F fibroblasts were assayed for L-dopa (Table 2). Only 208F/TH-8 cellscultured in media supplemented with 6MPH₄ produced L-dopa. Control cellsdid not contain any detectable amounts of L-dopa. Dopamine and itsmetabolites DOPAC and HVA were below detectable levels in both 208F/TH-8and 208F control cells.

                  TABLE 2                                                         ______________________________________                                        L-dopa Concentration of Cell Extracts and Media                                        L-dopa Concentration.sup.1                                                    Cell Extract Cell Media                                              Cell Clone no 6MPH.sub.4.sup.2                                                                     +6MPH.sub.4.sup.3                                                                      no 6MPH.sub.4.sup.2                                                                   +6MPH.sub.4.sup.3                       ______________________________________                                        208F/CONTROL                                                                             <0.25     <0.25    .sup. N.D..sup.4                                                                       63                                     208F/TH-8  <0.25      1.38    N.D.    239                                     ______________________________________                                         1. Ldopa concentration is exdpressed in units of nanograms (ng)/mg protei     for cell extracts and in units of ng/hr/10.sup.6 cells for cell media.        2. Cell incubated in normal media.                                            3. Cells incubated overnight in normal media supplemented with 0.1 mM         DL6-Methyl-5,6,7,8-tetrahydropterin.                                          4. N.D. not determined                                                   

As shown in Table 2, L-dopa was also detected in the media of the208F/TH-8 cells: 63 ng/hr per 10⁶ cells in control 208F media and 239ng/hr per 10⁶ cells in TH-infected media. There was no detectable levelsof L-dopa metabolites contained or released in 208F/TH8 or controlfibroblasts.

Histologic Examination of Grafts

Fibroblast grafts survived intraparenchymal transplantation to manyareas within denervated caudate. Surviving fibronectin positive graftswere typically moderate to large in size regardless of placement (FIG.18a and b). Only 4 out of 31 grafts were classified as non-survivingbased on the confinement of fibronectin staining to the syringe tract(FIG. 18c and d). Behavioral data from the rats with nonsurviving graftswere excluded from statistical analyses. TH immunoreactivity was notobserved in the fibroblasts either in vitro or in vivo.

Effect of Grafts on Rotational Asymmetry

The number of drug-induced rotations for each individual animal werecompared before and 2 weeks after transplantation. Rotational scoresfrom rats tested with apomorphine were pooled with those from ratstested with amphetamine since no difference was seen between thesegroups.

Amelioration of rotational asymmetry was dependent on graft placement.Rats with fibroblast grafts confined to caudal areas of the caudate(FIG. 19 Top) (AP=0 to 0.4) had no significant changes in rotationalbehavior. Rats which had surviving TH-infected fibroblasts in rostralcaudate striatum (AP=1.4 to 2.2) showed an average 33% reduction indrug-induced rotations 2 weeks following transplantation (FIG. 19Bottom).

These results demonstrate that the rat cDNA coding for the TH gene canexpress functional TH enzymic activity when stably transduced intofibroblasts. Fibroblasts expressing the TH gene can produce and secreteL-dopa in vitro. When these DOPA-producing fibroblasts were implantedinto the rostral caudate region, they substantially and significantlyreduce the rotational asymmetry in the rat model of Parkinson's. Sincecontrol fibroblasts do not produce significant levels of L-dopa in vitroand do not attenuate the rotational asymmetry of these rats, the abilityof these DOPA-producing cells to attenuate these rat's rotationalsymmetry must be due solely to the presence of the TH gene within thecells. These data demonstrated an effect on rotational behavior for atleast two weeks.

The effect of the DOPA-producing fibroblasts on rotational behavior weredependent on placement in the rostral caudate. Previous data utilizingfetal neuronal grafts into rats have shown that attenuation ofrotational asymmetry is best achieved when the grafts are placed intothe rostral caudate, (Dunnett, supra). Since the fibroblasts used cannotsprout axons, the location of the graft is even more criticallydependant upon proper graft placement.

The exact mechanism by which the DOPA-producing fibroblasts reducerotational asymmetry remains to be determined. Presumably, once L-dopais secreted, there remains enough caudate DOPA decarboxylase activity,even within these totally denervated animals (Lloyd et al., Science 170:1212-1213 (1970); Hornykiewicz, British Med. J. 29: 172-178 (1973)), toconvert L-dopa to dopamine that then modifies drug-induced rotationalbehavior. This postulated mechanism of action of these DOPA-producingcells would be consistent with the well established efficacy of systemicL-dopa therapy for Parkinson's disease (Calne, N. Eng. J. Med. 310:523-524 (1984)). These DOPA-producing fibroblasts are in effect smalllocalized pumps of L-dopa.

The ability, demonstrated in this example, to modify cells to produceL-dopa broadens the search for the ideal type of cell fortransplantation therapy of Parkinson's. Any cell that can be geneticallymodified to express the TH gene and that can survive long-term in thebrain without forming a tumor or causing other damage, may be used.Although these particular immortalized rat fibroblasts have not formedtumors for up to three months, primary cells such as primary fibroblastsor primary glial cells may offer the theoretical advantage of decreasedpropensity for tumor formation and may be preferred for grafting incertain applications. In addition, the use of the patients own primarycells for an autologous graft would decrease the chance of graftrejection. However, the use of immortalized cells such as the 208F cellsused in this example, does offer the advantage of having large amountsof well-characterized cells readily available.

These results demonstrate that a fibroblast can be genetically modifiedto supply a function normally supplied by a neuron, therefore notrequiring the use of fetal tissue for neuronal transplantation. Theability to combine transplantation modalities with gene-transferpresents a powerful method for the treatment of CNS dysfunction. Themethods of the present invention may thus be used for treatment of othermodels of animal and human brain disease.

EXAMPLE IV Grafting of Cultured Autologous Primary Skin Fibroblasts

As noted above, it may be desirable in certain circumstances to minimizerejection of intracerebral grafts and avoid tumor formation usingautologous cells such as primary skin fibroblasts. In this example,primary skin fibroblasts were used to evaluate this cellular populationfor intracerebral grafting by assessing the growth and survival of thecells in vitro and in vivo and the integration of the grafted cells intothe donor's striatum.

In these experiments, the growth rate of primary skin fibroblasts inculture was measured. This growth pattern was compared with that ofimmortalized Rat-1 fibroblasts in vitro, to determine whether growth andcellular characteristics in culture would predict those followingimplantation. Variables that might influence the survival of primaryfibroblast grafts were also examined, including the number of passagesthe cells can be taken through in culture before implantation and thedensity of cells implanted. Introduction of new genetic material intocultured cells requires several passes to select and expand the modifiedpopulations, making assessment of the former parameter important. Thedensity of cells implanted was varied to determine an optimal graftsize. The morphological and neurochemical characteristics of the graftswere also examined, and the cellular interactions between the autologousfibroblast grafts and host striatum were revealed.

Skin biopsies were taken from the ventral abdominal wall of twenty-threefemale Sprague-Dawley rats (200-250 g). Pure fibroblast cultures fromeach biopsy were maintained under standard conditions and fed DMEMcontaining 10% fetal bovine serum three times a week. The fibroblastswere grown to confluency and then split 1:2. The growth rate of primaryskin fibroblasts was determined with the aid of a Coulter Counter(Coulter Electronics Inc). Triplicate samples of these cells werecounted each day, and the growth rate of primary fibroblasts wascompared to that of immortalized Rat-1 fibroblasts grown and sampledunder the same conditions.

For intracerebral grafting, primary skin fibroblasts from each animalwere taken to either one or four passages (there were approximately fivecell divisions between passage one and four). These cells were thengrown to confluency and harvested at a resting state. The culturedfibroblasts were suspended in a solution of grafting phosphate bufferedsaline supplemented with 1 mg/ml of MgCl₂ and CaCl₂, and 0.1% glucose.The density of the suspensions ranged from 10⁴ to 10⁵ cells/μl. Theanimals were anesthetized with intramuscular injections ofketamine-xylazine (10 mg/kg Ketarar, Parke-Davis, Ann Arbor, Mich.; 5mg/kg Rumpun, Hoechst, Frankfurt, Germany; and 6 mg/kg acepromazinemaleate, TechAmerica) and placed in a stereotaxic frame. Each animalreceived implants of cultured skin fibroblasts taken from its own skinbiopsies. A total of four cell suspensions (3 μl/site) were injectedstereotaxically into the striatum with a Hamilton syringe, two rostrallyand two caudally. Following implantation, the animals survived forperiods of three or eight weeks.

To assess parameters that may influence the survival of intracerebralprimary skin fibroblast grafts, two experiments were designed to addressthe issues of cell passage (n=12) and cell density (n=9). First, groupsof three rats received injections of their own primary skin fibroblasts(10⁵ cells/μl) that were taken through one or four passages; theseanimals were sacrificed and perfused three or eight weeks followingsurgery. Second, groups of three animals received suspensions at 10⁴,2.5×10⁴, 5.0×10⁴ or 10⁵ cells/μl; the number of passages (four) and thesurvival period (eight weeks) were constant.

Tissue Preparation and Histology

For light microscopy, the animals were perfused transcardially with asolution of 4% paraformaldehyde in phosphate buffer (pH 7.3). The brainswere removed, post-fixed overnight, and stored in 30% sucrose for threedays. Coronal sections through the striatum were cut on a freezingmicrotome at a thickness of 40 μm and collected in cryoprotectant. Oneseries of sections was stained with cresyl violet. The remainingsections were divided into three groups for the immunohistochemicaldetection of fibronectin, glial fibrillary acidic protein (GFAP) andreactive microglia and/or macrophages. The sections were treatedinitially with 0.6% hydrogen peroxide in Tris buffer (pH 7.4) for thirtyminutes to block endogenous peroxidase activity. Sections were rinsed inbuffer and then incubated in rabbit anti-human fibronectinimmunoglobulin (IgG) (1:2000 dilution) or rabbit anti-human GFAP IgG,1:1000 dilution) with 3% normal goat serum and 0.25% Triton-X in Trisbuffer overnight at room temperature. Next they were rinsed in bufferand incubated in biotinylated goat anti-rabbit IgG (1:250 dilution) forone hour. After another rinse, they were incubated in avidin-biotincomplex (Vector Laboratories) for one hour and rinsed. They were thentreated with a solution of 0.025% diaminobenzidine (DAB)tetrahydrochloride, 0.5% nickel chloride and 0.03% hydrogen peroxide inTris buffer for five minutes.

Immunohistochemical localization of reactive microglia and/ormacrophages was achieved using the mouse monoclonal antibody MRC OX-42(Serotec). Sections were rinsed in buffer, and then incubated with MRCOX-42 (at a concentration of 10 μg/ml) with 3% normal horse serum and0.25% Triton-X in Tris buffer overnight at room temperature. They wererinsed and incubated in biotinylated horse anti-mouse IgG (1:160dilution) for one hour. The sections were then rinsed, incubated inavidin-biotin-complex, rinsed again, and reacted in a DAB solution (asabove). Following the immunoperoxidase reaction, all sections wererinsed in Tris buffer and then in phosphate buffer. They were mounted onchrome alum-gelatin coated slides, dehydrated through a graded series ofethanols, and coverslipped.

For electron microscopy, animals (n=2, from the cell density study) wereperfused transcardially with a solution of 4% paraformaldehyde and a0.1% glutaraldehyde in Tris buffer (pH 7.4). The brains were removed andpost-fixed for three hours, after which coronal sections through thestriatum were cut on an Oxford vibratome at a thickness of 50 μm andcollected in Tris buffer. Sections were then either stainedimmunohistochemically for fibronectin or fixed in 1% buffered osmiumtetroxide for one hour. The latter sections were rinsed, dehydratedthrough a graded series of methanols, cleared in propylene oxide, andembedded in a mixture of Poly/Bed and Araldite. Semithin sections (1-2μm) were collected and stained with 1% toluidine blue to assess thegraft. Ultrathin sections were then cut on a Reichert Om U3ultramicrotome, collected on copper grids and stained with uranylacetate and lead citrate. The sections were viewed with a JEOL 100 CXIItransmission electron microscope.

Graft Analysis

Since fibronectin is not normally detected in the rat brain, fibroblastgrafts were defined by the area stained immunohistochemically forfibronectin. Sections possessing immunoreactivity were analyzed with aCue-2 Image Analysis System (Olympus Corp). Immunostaining was tracedmanually (mag.=4X) and the area of the graft was computed for eachsection in a given series. Graft volume (mm³) was calculated from thisseries according to Uylings et al. (J. Neurosci. Meth. 18: 19-37(1986)), incorporated by reference herein. Sections stained for cresylviolet, GFAP and MRC OX-42, as well as sections viewed with the electronmicroscope, were evaluated qualitatively.

Growth Curves for Rat-1 Fibroblasts and Primary Fibroblasts

The growth rate of Rat-1 cells, which are immortalized fibroblasts thatare putatively contact inhibited, was compared to that of primary skinfibroblasts cultured under similar conditions. Although the growthcurves of both cell types were similar for the first week, dramaticallydifferent growth patterns were observed after ten days (FIG. 20). Rat-1cells continued to grow once confluency was reached, and therefore didnot appear to be contact inhibited. The growth of primary skinfibroblasts, on the other hand, stabilized once the cells reachedconfluency. These patterns of continued growth (Rat-1 fibroblasts)versus quiescence (primary skin fibroblasts) persisted for up to threeweeks.

Cell Passage

A parameter that may influence the size of primary fibroblast grafts inthe striatum was the number of in vitro passages the cells were takenthrough prior to implantation. For this study, the cell density ofgrafts was constant at 10⁵ cells/μl. Six animals received cellsuspensions of fibroblasts taken through one passage in culture, andanother six received fibroblasts taken through four passages. Threeanimals from each group were sacrificed and perfused three or eightweeks after implantation. A 2-factor ANOVA was employed to assess thedifference in volume of grafts at passage one and four, and afterpostoperative periods of three and eight weeks (FIG. 21). Graft volume,determined with fibronectin immunostaining, did not differ as a functionof the number of passages in culture prior to implantation (one versusfour). It was also evident that cultured autologous fibroblastsimplanted into the brain did not create tumors, nor did these graftsatrophy and die. In fact, the size of the grafts did not differsignificantly after three and eight weeks survival in vivo.

Cell Density

Another parameter of graft size examined was the density of cellsimplanted into the striatum. Four densities (10⁴, 2.5×10⁴ and 10⁵cells/μl) were assessed, and the volume of the grafts was determinedimmunohistochemically for fibronectin. The fibroblasts for intracerebralgrafting were harvested following the fourth passage in culture and theanimals were sacrificed after a post-operative period of eight weeks.With a 2-factor ANOVA, significant differences in graft volume wereobserved among the fours groups; suspension grafts of high cell densityproduced larger grafts than grafts of low cell density (FIG. 22). Thus,a relationship between the volume of the graft and the density of cellsimplanted was evident.

Histology

Immunostaining for fibronectin, a group of structurally relatedglycoproteins, revealed the size and limits of the intracerebral grafts.At three weeks, the grafts possessed several small extensions into theneuropil of the striatum, whereas those examined at eight weeks appearedmore compact and possessed fewer projections (FIG. 23). Fibronectinimmunostaining, however, did not allow for the resolution of individualfibroblasts or other cellular components in or near grafts. From cresylviolet staining, elongated nuclei that are typical of fibroblasts wereobserved in the grafts (FIGS. 24a, b). Blood vessels were also foundthrough the grafts, and debris-filled cells were located predominantlyat the center. Astrocytes stained immunohistochemically for GFAP wereprominent in the host striatum surrounding the grafts at three weeks,yet immunostaining within the grafts was sparse. GFAP-immunoreactiveprocesses were observed both within and around the grafts at eight weeks(FIGS. 24c, d). Reactive microglia and/or macrophages visualized withMRC OX-42 immunoreactivity surrounded the grafts at three weeks, butwere less prevalent and more variable at eight weeks (FIGS. 24e, f).

Ultrastructure

The structural organization of primary fibroblast autografts at eightweeks after implantation was examined at the electron microscope level.The most striking feature of the grafts was the abundance of fibroblastsand collagen. The fibroblasts possessed an elliptical nucleus that wasoften condensed; a distinct nucleolus was rarely observed (FIG. 25a, b).The cytoplasm of these cells contained rough endoplasmic reticulum,elongated mitochondria, and secretory vesicles. a paucity of Golgiapparatus was evident. Slender fibroblast processes found throughout thegrafts also contained rough endoplasmic reticulum and secretory vesicles(FIG. 25c). Dense bundles of collagen surrounded fibroblast cell bodiesand their processes. Neither fibroblasts nor collagen were observed inthe striatal neuropil. Other cellular components of the grafts includedreactive astrocytic processes and phagocytes. Hypertrophied astrocyticextensions were observed in the extracellular matrix of the graftspassing among collagen bundles; the cell bodies of astrocytes were notfound within the fibroblast implants (FIG. 26a). The morphology ofphagocytic cells of the grafts was variable; the cytoplasm possessed feworganelles and was usually filled with electron-dense debris (FIG. 26b).The nucleus of these cells was irregular and chromatin was often clumpedbeneath the nuclear envelope. A small number of lymphocytes was alsoobserved around and within the grafts at eight weeks after grafting.

Another prominent feature of the primary fibroblast grafts was thepresence of continuous-type capillaries; the endothelial lining of thelumen was uninterrupted, and fenestrations were not evident (FIG. 27a).A basal lamina enveloping these vessels was present. Endothelial cellssurrounding the lumen possessed few organelles and a nucleus withcondensed chromatin under the nuclear envelope. Numerous vesicularinvaginations of both the luminal and abluminal plasma membranes ofendothelial cells were evident, as well as clear vesicles within thecytoplasm (FIG. 27b). Intercellular junctions were observed at sites ofclose apposition between endothelial cells (FIG. 27c). Collagen and/orreactive astrocytes were closely associated with the graft capillaries.These reactive astrocytic profiles adjacent to capillaries were filledwith filaments, and did not resemble those endfeet astrocytes thatenvelope normal cerebral vessels (FIG. 27d). Certain capillaries alsopossessed several reactive astrocytic processes completely surroundingthe vessel; the perivascular space between the endothelium andastrocytes usually possessed collagen and was variable in size.

Primary skin fibroblasts cease to proliferate once they are confluent,thereby exhibiting contact inhibition in vitro. As shown herein,following implantation in the striatum, the graft volume of primary skinfibroblast grafts remains constant between three and eight weeks; thus apropensity for either tumor formation or death is not a characteristicof primary skin fibroblasts in vivo. Features of primary fibroblasts inculture, e.g., contact inhibition, may reflect the growth and survivalof the grafts following implantation into the CNS. Data from the presentinvestigation also reveal that the density of the cell suspension isanother factor that determines the size of primary skin fibroblastgrafts: cell suspensions of a high density produce larger grafts thanthose of a low density. The number of cell passages in culture prior toimplantation and the post-operative period, on the other hand, do notappear to be parameters that influence graft size.

Another advantage of employing primary skin fibroblasts for grafting isthat the likelihood of immune rejection of autologous cells is lessened.An immunological response against allografts, especially 208Ffibroblasts, may be one factor that leads to cell death followingimplantation. In this example, fibroblasts from skin biopsies were grownunder cultured conditions and implanted into the donor's striatum.Following stereotaxic placements of fibroblasts in the striatum, theblood-brain barrier is damaged, and several cell populations, includinglymphocytes, enter the wound area. Despite the presence of lymphocytesnear the graft, the immunological reaction against these autologouscells appears to be minimal. Therefore, the histocompatibility ofprimary fibroblasts may also enhance graft survival in vivo.

This example provides direct evidence for the sustained survival ofprimary skin fibroblasts grafted in the adult rat CNS. Ultrastructuralanalysis reveals that these grafts are composed primarily of fibroblastsand collagen, in addition to possessing other cellular populations(e.g., phagocytes, lymphocytes). It is noteworthy that the morphologicalfeatures of grafted fibroblasts after eight weeks in the striatum aresimilar to those normally found in the skin, including an ellipticalnucleus with condensed chromatin, elongated mitochondria, and a fewGolgi apparatus. The presence of rough endoplasmic reticulum andsecretory vesicles within the cytoplasm and processes of the fibroblastsstrongly indicates active protein production. Further, the abundance offibronectin immunostaining and collagen provide evidence for thesynthesis of two distinct cellular products of fibroblasts within thegrafts. Despite collagen production, the size of the grafts remainsrelatively constant between three and eight weeks after implantation.One plausible explanation for this observation is that culturedfibroblasts are known to produce collagenase (Millis et al., Exp.Geront. 24: 559-575 (1989)); fibroblasts may therefore regulate thelevels of collagen production and degradation within the grafts. Fromthe light and electron microscopic date, it is concluded that primaryskin fibroblasts grafted in the rat striatum maintain theirmorphological characteristics, produce and secrete collagen andfibronectin, and survive up to eight weeks. Although the total number ofsurviving cells or the percentage of cells that survive was notdetermined, a higher percentage of cells within the grafts at eightweeks following implantation is unlikely. Trophic substances such asfibroblast growth factor (FGF) may augment the survival of primary skinfibroblasts grafted into the rat CNS.

Host-to-graft Interactions

An important consideration of cultured autologous skin fibroblastimplants is whether these grafts are integrated into the host nervoussystem. After implantation of cultured primary skin fibroblasts into therat striatum three prominent cellular events were observed thatconstitute dynamic interactions between the host and graft. These may betemporal events and occur as follows: 1) activation and migration ofmicroglia and/or macrophages to the graft; 2) neovascularization; and 3)hypertrophy and infiltration of astrocytic processes into the graft.Data from prior investigations of the initial response to the CNS topenetrating wounds that compromise the blood-brain barrier may beextrapolated to the model system described herein. It has been shownthat mononuclear phagocytes are the first cells that respond to woundswithin the CNS (Imamoto and Leblond, J. Comp. Neurol. 174: 255-280(1977)). Macrophages, microglia and granulocytes may be shownimmunohistochemically using the MRC OX-42 antibody which recognizes therat homologue of complement receptor type 3 of mouse and man (Robinsonet al., Immunol. 57: 239-247 (1986)). The results presented hereinindicate that cells possessing OX-42 immunoreactivity are present at theperiphery of the graft at three and eight weeks after implantation.Electron microscopic examination of tissue at eight weeks confirms thepresence of phagocytic cells in the interface zone between the graft andthe intact neuropil, and these cells may be those stainedimmunohistochemically for OX-42. Phagocytic cells were also presentthroughout the graft, yet OX-42 immunoreactivity was not evident withinthe graft itself. Therefore, non-immunoreactive phagocytes mayconstitute either another cellular population or macrophages, microgliaand granulocytes that lack the complement receptor. The cells migrate tothe wound site and infiltrate the graft.

An important host-to-graft cellular interaction that may determine thefate of fibroblasts following implantation is the establishment of avascular system within the grafts. The establishment of a vascularsystem within the grafts may depend in part on angiogenic substancessuch as fibroblast growth factor. Production and release of fibroblastgrowth factor by grafted skin fibroblasts may promote angiogenesis.Neovascularization within the grafts may also be enhanced by reactivemicroglia and macrophages which produce and release interleukin-1.

Implants of avascular tissue into the CNS are completely dependent onthe surrounding host tissue for the formation of new capillaries.Capillaries within the skin fibroblast grafts are composed ofnon-fenestrated endothelial cells that form a continuous lumen andpossess intercellular junctions (not necessarily tight junctions) atsites of cellular apposition; these vessels are reminiscent of thosefound in both connective and nervous tissues; however several featuresdistinguish graft capillaries from those in the adjacent striatum.First, endothelial cells within the grafts possess numerousintracellular vesicles and vesicular invaginations on the plasmamembranes, whereas neural capillaries have few endothelial invaginationson the luminal and abluminal sides. Second, reactive astrocytesassociated with graft vessels are unlike endfeet astrocytes surroundingneural capillaries, in that the former profiles are filled withfilaments, hypertrophied and do not always envelope the graftcapillaries. Third, the perivascular space between endothelium andreactive astrocytic profiles of graft capillaries is variable and oftencontains intervening elements, including collagen; neural vesselspossess a narrow perivascular space containing basal lamina.

The third interaction observed between the striatal neuropil and graftsof primary fibroblasts was the responsiveness of astrocytes to theautografts. Intracerebral grafting usually results in a glial reactionthat involves the migration of reactive astrocytic processes and/or theformation of a glia limitans. These studies show thatGFAP-immunoreactive astrocyte perkarya are abundant around the graft atthree and eight weeks, and immunoreactive processes are found within thegraft at eight weeks. Ultrastructural data corroborate the latterobservation, revealing that reactive astrocytic processes are presentthroughout the graft, but astrocytic cell bodies remain outside thegraft itself. Astrocytic responses observed in the present study andothers may be due, in part, to the presence of reactive microglia andmacrophages near the wound site; these cells are known to produce andrelease interleukin-1 in vitro.

These results provide direct evidence for the survival of culturedprimary skin fibroblasts grafted into the CNS of adult rats. Thesegrafts do not form tumors within the striatum, and possibleimmunological responses appear minimal due to the autologous nature ofthese cells. Moreover, fibroblast grafts are structurally andfunctionally integrated into the host brain. Dynamic cellularinteractions between the host striatum and fibroblast implantsindicating the structural and functional integration of the fibroblastsinto the adult rat CNS include the establishment of a vascular systemwithin the graft, the migration of phagocytic cells, and the hypertrophyand infiltration into the grafts of reactive astrocytic processes.Collectively, these features indicate that primary skin fibroblasts aresuitable donor cell candidates for cerebral grafting, and therefore maybe employed as genetically modified cells for gene therapy in diseasedor damaged CNS.

EXAMPLE V Grafting of Primary Fibroblasts Producing L-dopa

This example examines the long term survival of genetically modifiedprimary fibroblasts following grafting into the brain. Extending theabove experiments, the rat TH gene was inserted into primary fibroblastsobtained from a skin biopsy from Fischer rats. The survival ofTH-containing primary fibroblasts was then examined for a 2 month periodfollowing implantation into the brain of Fischer rats with a prior6-OHDA lesion of the nigrostriatal pathway. In addition, both thepresence of the TH transgene and transgene product within the implantedfibroblasts were examined. Finally, prolonged functioning of thegenetically modified fibroblasts in vivo was explored through assessmentof rotational behavior of the implanted rats every 2 weeks for an 8 weekperiod after grafting.

Preparation of Retroviral Vector

Preparation of the vector used in the present experiments, pLThRNL, wasas described above in Example III (see FIGS. 16 and 17).

Fibroblast preparation

Primary fibroblasts were obtained from abdominal skin biopsies of inbredFischer 344 rats, according to the protocol of Sly and Grubb (in"Methods in Enzymology", Vol. LVIII, Colowick and Kaplan, Eds., AcademicPress, New York, pp. 444-450 (1979)). Briefly, rats were anesthetizedwith a mixture of ketamine, aceptomazine and rompun. The abdomen wasshaved and scrubbed thoroughly with alcohol. An area of approximately1-2 cm² of skin was removed, rinsed briefly in alcohol and immediatelyplaced in a vial containing sterile DMEM/S. Biopsies were typicallytransferred to coverslips within 2 hours of collection and grown inDMEM/S for 21 days. Cells were then infected with ecotropic LThRNL orBAG virus (Price et al. Proc,. Natl. Acad. Sci. USA 84: 156-160 (1987))at a multiplicity of infection of 10 in the presence of Polybrene (4μg/ml). G418-resistant cells were grown to confluency and tested forexpression of TH activity by a decarboxylase-coupled assay (Iuvone, J.Neurochem 43: 1359-1368 (1984)). A subclone expressing TH activity andTH immunoreactivity in vitro was selected for implantation (FF2/TH).Fibroblasts infected with the BAG virus and expressing β-galactosidase(βGal) activity in vitro were used as control cells (FF2/βGal).

In vitro Biochemical Analyses

The production and release of L-dopa and its metabolites from theinfected fibroblasts were assessed after growing cells for 24 hours inDMEM/S supplemented with 100 μm tetrahydrobiopterin, the active cofactorfor TH. Conditioned media and cells were collected, adjusted to 0.1Mperchloric acid (PCA) and 0.05M EDTA, and centrifuged at 10,000 X g for15 min at 4° C. to remove precipitated material. Samples were analyzedfor the presence of catecholamines and catecholamine metabolites asdescribed above in Example II by injecting the PCA extracts onto acoulometric electrode array, gradient liquid chromatography system (CEASmodel 55-0650, ESA, Bedfore, Mass.) equipped with 16 electrochemicalsensors (Matson et al., Clin. Chem. 30: 1477-1488 (1984); Langlais etal., in "Monitoring Neurotransmitter Release During Behavior", Fillenz,et al., eds., Horwood, Chichester, U.K., pp. 224-232 (1986); and Matsonet al., Life Sci. 41: 905-908 (1987)).

Lesions

The dopaminergic nigrostriatal pathway was unilaterally destroyed withthe neurotoxin 6-hydroxydopamine (6-OHDA). The 6-OHDA was dissolved insaline at a concentration of 6 μg/μl and supplemented with 0.1% ascorbicacid to retard oxidation. Female Fischer 344 rats (130-160 g) wereanesthetized as above and placed in a stereotaxic frame. A 2 μlinjection of 6-OHDA was delivered at 1 μl/min to the left medialforebrain bundle (AP=-4.4 mm, ML=1.1 mm, DV=7.5 mm; according to theatlas of Paxinos and Watson, 1986, "The Rat Brain in StereotaxicCoordinates", San Diego, Academic Press, (1986)). Following theinjection, the syringe was raised 2 mm and allowed to rest another 2minutes to permit diffusion of the neurotoxin. After a 10 to 14 dayrecovery period, lesioned rats were tested with apomorphine to assessthe extent of the lesion.

To assess the contribution of non-specific striatal damage to changes inrotational behavior the procedures described above in Example III wereemployed using 6-OHDA lesioned rats subjected to unilateral kainic acid(KA) lesion of the striatum. For this experiment, female Sprague-Dawleyrats were anesthetized and lesioned as above with 6-OHDA. Rats whichdisplayed greater than 7 rotations per minute to apomorphine (0.05mg/kg) were selected for KA lesion. Kainic acid was dissolved in salineat a concentration of 5 μg/μl and injected into the striatum (AP=0.3 mm,ML=2 mm, DV=4 mm) of anesthetized rats at varying doses. Three ratsreceived 1.0 μg, 3 rats received 2.0 μg, 3 rats received 4.0 μg and 2rats received 5.0 μg. The KA was delivered at a rate of 0.1 μl/min, andthe syringe was then slowly lifted 2 mm and left in place for anadditional 2 minutes to allow diffusion of the neurotoxin. Rats wereadministered diazepam (2 mg/kg) immediately following the injection toblock seizures which may have resulted from the use of the KA.

Behavioral Testing

The sensitivity of postsynaptic dopamine receptors within the denervatedstriatum was assessed 7-10 days after 6-OHDA lesion with apomorphine(0.1 mg/kg) administration as described above in Example III. Rats wereplaced in automated rotometer bowls and total rotations were collectedevery 10 minutes for a period of 60 minutes. After 2 apomorphine tests,rats which displayed at least 7 rotations/minute for 60 minutes wereselected for further study. Rats were tested with apomorphine at least 3times or until the number of rotations did not change from the previoustesting session (Bevan, Neurosci. Letter 35: 185-189 (1983); Castro etal., Castro et al., Psychopharm. 85: 333-339 (1985)). A period of 4-10days elapsed between each apomorphine test. After grafting, rats weretested with apomorphine (0.1 mg/kg) every 2 weeks for 8 weeks. Rats withKA lesions were tested with apomorphine (0.05 mg/kg) 2, 4 and 6 weeksafter lesioning. For statistical comparisons, the net rotations/minuteover a 60-minute time period were determined following grafting andcompared to pre-transplantation values using the Least SquaresDifference Test.

Grafting

Fibroblasts were prepared for grafting as described above in Example II.Briefly, cells were removed from tissue culture plates with 0.05%trypsin and 1 mM EDTA in Dulbecco's PBS, and suspended in PBSsupplemented with MgCl₂, CaCl₂, 0.1% glucose (complete PBS) and 5% ratserum. Cells were collected by centrifugation, washed twice withcomplete PBS, and resuspended in complete PBS at a density of 100,000cells/μl.

Fifteen anesthetized rats received a total of 10 μl of either FF2/TH(N=10) or FF2/βGal (N=5) fibroblasts injected into two sites within thestriatum (AP=0.3 mm, ML=2.0 mm, DV=4.5 mm, AP=1.5 mm, ML=2.0 mm, DV=4.5mm). At each site, 2.5 μl of cells were dispensed, the syringe wasraised 1 mm, and an additional 2.5 μl of cells were ejected from thecannula. Cells were delivered at a rate of 1 μl/min. After the mostdorsal injection, the syringe was left in place for 2 minutes to allowdiffusion of the cells. An additional 5 lesioned rats, which were notgrafted, served as untreated controls.

Anatomical Procedures

For in vitro immunohistochemical analyses, infected fibroblasts werefixed onto slides with 4% paraformaldehyde, permeabilized with 0.2%triton X-100 and incubated overnight with a monoclonal antibody to TH(Boehringer Mannehim Biochemicals). The stain was developed by theavidin-biotin method (Vector Laboratories, Elite Kit) withnickel-intensified 3,3'-diethylaminobenzidene (DAB) as the chromagen.

For in vivo analyses, implanted fibroblasts were examined for TH- andfibronectin-immunoreactivity 10 weeks after grafting. Rats were deeplyanesthetized and perfused with 4% paraformaldehyde following a briefsaline rinse. Brains were removed and placed in the same fixativeovernight and then transferred to 30% sucrose until they equilibrated.Frozen sections were cut on a sliding microtome (40 μm) and collected incryoprotectant for storage until processing. Free floating sections wereincubated overnight with either an antibody to the fibroblast marker(Organon Technica), or with the monoclonal antibody to TH. Fibronectinstaining was developed with avidin-biotin followed by nickle-intensifiedDAB. TH labeled sections were incubated with a secondary antibody to themouse antigen conjugated to rhodamine for fluorescence microscopy(Jackson ImmunoResearch Labs., Inc., Bar Harbor, Me.). Some sectionswere also processed with antibodies OX-42 (Serotec) or ED-1 (Chemicon)for identification of microglia or monocyte/macrophages. Sections wereincubated with the OX-42 antibody for 48 hours and the ED-1 antibody for24 hours at 4° C. The following day, OX-42 was developed withavidin-biotin with nickle-intensified DAB while the ED-1 labeledsections were incubated with the rhodamine-labeled secondary antibody asdescribed above.

In situ Hybridization

In situ hybridization for TH mRNA was performed using [³⁵ S] labelledprobes as described by Higgins et al., Proc. Natl. Acad. Sci. (USA) 85:1297-1301 (1988), incorporated by reference herein. A 63 base pairtemplate for the TH probe was generated by digestion of the plasmidpSP65 containing a 311 nucleotide cDNA complementary to rat TH mRNA(provided by Dr. Dona Chikaraishi, Tufts Medical School, Boston, Me.)with Ava II, and transcribed by the SP6 polymerase. Slide-mountedsections were fixed for 1 minute with 4% paraformaldehyde, digested withproteinase K for 5 minutes and then incubated in triethanolamine (0.1 M,0.9% NaCl and 0.1% acetic anhydride) for 10 minutes at room temperature.Sections were subsequently dehydrated in graded alcohols followed bychloroform for 5 minutes at room temperature, returned to 100% ethanolfor 2 minutes and then 95% ethanol for another 2 minutes prior to airdrying. Dehydrated sections were prehybridized for 1-3 hours in buffercontaining 50% formamide, 0.75M NaCl, 20 mM Pipes (pH 6.8), 10 mM EDTA,250 mMDTT, 5X Denhart's solution (0.02% BSA, 0.02% Ficoll, 0.02%polyvinylpyrrolidone), 0.2% SDS, 10% dextran sulfate, and 500 μg/mldenatured yeast tRNA. Following prehybridization, excess buffer wasremoved and sections were hybridized at 55° C. for 18 hours with 10-15ng of labeled probe in a total volume of 75 μl of hybridization buffer.Slides were thoroughly rinsed following the hybridization in solutionscontaining 0.6M NaCl, 0.06M Na citrate with or without RNase. Followingposthybridization rinsing, the sections were air-dried and exposed toX-ray film (Kodak XAR) for 24 hours. The sections were then dipped inKodak NTB-2 emulsion, exposed for 4 days (at 4° C.), and developed.

When the bulk population of TH-expressing fibroblasts was stained forTH-immunoreactivity, differing intensities of TH-labeling were observed.To try to isolate a clone that stably expressed high levels of TH, thebulk population of TH-expressing cells was subcloned. The cloneselected, FF2/TH, exhibited a relatively homogeneous TH-immunoreactivity(FIG. 28). This clone was similar in gross morphological appearance touninfected fibroblasts or fibroblasts infected with the vector carryingβGal. Cells were generally elongated with multiple processes extendingfrom the soma. Cells expressing βGal did not display TH-immunoreactivityin vitro.

In Vitro Analyses

Primary fibroblasts infected with vectors carrying genes for TH or βGal(FF2/βGal) were examined for TH activity using a decarboxylase-coupledassay. TH activity was only found in FF2/TH cells, at a level of 1.1±0.4pmole L-dopa/minute/mg protein. This level of TH within the FF2/THprimary fibroblasts was comparable to that seen previously forimmortalized 208F fibroblasts infected with the same LThRNL vector (seeExample III above).

Infected fibroblasts were assayed for the production and release ofL-dopa and other catecholamines 24 hours after exposure to 100 μM BH₄,the natural cofactor for TH. Medium collected from cells containing theTH gene was found to contain 17.2 μg L-dopa/10⁶ cells. Intracellularquantities of L-dopa, as assessed through analysis of the FF2/TH cellpellet, were 0.2 μg L-dopa/10⁶ cells. Control FF2/βGal cells did notcontain or release L-dopa. There were no detectable metabolites ofL-dopa, such as dopamine, 3,4-dihydroxyphenylacetic acid or homovanillicacid, in samples collected from the media or cell pellet of eitherFF2/TH or FF2/βGal cells.

Behavioral Analyses-fibroblast implanted rats

All 6-OHDA lesioned rats implanted with FF2/TH or FF2/βGal cells wereeating and drinking normally within 24 hours of the transplantationsurgery and showed stable or slightly increasing body weight during the2-month post-grafting test period. There was no obvious change inspontaneous behavior of the grafted rats; grooming and locomotorbehaviors were comparable to those displayed by the lesioned controlrats that Were not grafted.

To assess the production and release of L-dopa from the implanted cellsin vivo, a behavioral test was conducted. Rats were administeredapomorphine, which results in activation of receptors in thedopamine-depleted striatum. This drug-induced stimulation causes therats to walk in a circular or rotational pattern which can be quantified(Ungerstedt and Arbuthnott, Brain Res. 24: 485-493 (1970)). Followinggrafting, average rotations per minute over a 60 minute period followingapomorphine administration were determined for grafted and controlnon-grafted rats and compared to pretransplantation rotations forstatistical analyses. Control animals showed a slightly increased levelof rotations following grafting (FIG. 29, control) which was notstatistically different from that observed prior to grafting (p>0.05).The apomorphine-induced rotations of rats implanted with βGal-containingcells were also not different from pregrafting rotations through the2-month post-grafting time period (p>0.05; FIG. 29, FF2/βGal). Incontrast, FF2/TH rats displayed a significant decrease in rotationalbehavior 2 weeks after grafting, which persisted through 8 weekspost-implantation (FIG. 29, FF2/TH). The initial 65% decrease inrotations of FF2/TH rats, from 11.7±3.0 to 4.1±2.9 (mean±standard errorof the mean), narrowed to a plateau of approximately 30% belowpre-grafting levels between 6 and 8 weeks post-grafting.

Anatomical Analyses

Fibroblast grafts were present within the brains of all of the implantedrats 10 weeks post-implantation. However, grafts from 2 animals werelost during processing. In general, the placement of FF2/TH and FF2/βGalgrafts were found to be similar, both extended along comparablerostral-caudal and medio-lateral domains. Typically, however, FF2/THgrafts were larger than FF2/βGal grafts. Within the FF2/TH group, therewas no correlation between the size of the grafts and reduction ofrotational behavior.

All grafts contained elongated fibroblast-like cells interspersed amongan abundance of collagen fibers. In addition to typical fibroblasts,large yellow cells, apparently filled with hemasiderin, were seen withinthe core of the grafts. As these cells were often filled with debris,sections through the graft were stained with OX-42, and ED-1, markersspecific for microglia and monocyte/macrophages. However, whileimmunoreactive macrophages were found on the periphery of the grafts,the large debris-filled core cells did not label with 0X-42 or ED-1.

To assess whether the TH transgene continued to be expressed within theimplanted fibroblasts, sections through the graft were processed for insitu hybridization to TH mRNA. Positive hybridization for TH mRNA wasonly found to be present over cells in the grafts containing FF2/THfibroblasts (FIG. 30a). In both FF2/TH and FF2/βGal grafts (FIG. 30b,control), areas of the graft which were predominantly composed ofcollagen fibers showed a grain density which was significantly less thanthe surrounding background. The morphology of the cells displayingincreased grain density was varied, with many exhibiting the elongatedshape characteristic of fibroblasts (FIG. 31c). However, some cells withpositive TH hybridization were observed to be large and rounded, oftenfilled with hemasiderin and granulated particles. There were no cellspresent within βGal grafts which displayed specific hybridization to THmRNA (FIG. 31d).

To determine if the transgene products were synthesized and presentwithin the fibroblasts, grafts were examined for TH-immunoreactivity andβGal histochemistry. Within the FF2/TH grafts, TH-labeled cells with theelongated shape typical of fibroblasts were observed to be scatteredamong the collagen fibers (FIG. 32a). Autofluorescent material waspresent within the grafts but was readily discernible form therhodamine-specific labeling (emission 582 nm) by the shape of thematerial and its broad fluorescence through 528 nm (FIG. 32a and 32b,asterisks). There was a heterogeneity in the intensity of the THlabeling, as observed in vitro. Autofluorescent cells, but nodiscernible TH-immunoreactive fibroblasts, were present within FF2/βGalgrafts.

Sections stained for βGal histochemistry revealed diffuse cytoplasmiclabelling of cells within the FF2/βGal grafts (FIG. 31a). FF2/βGalstaining was observed in some of the large rounded cells within the coreof the graft and in cells with the elongated shape typical offibroblasts (FIG. 31b). βGal reactivity within FF2/TH grafts wasconfined to a small number of rounded cells with the blue granularstaining previously described for endogenous macrophages (Shimohama etal., Mol. Brain Res. 5: 271-278 (1989)).

These results confirm that primary fibroblasts obtained from a skinbiopsy can be grown and genetically manipulated in culture to express atransgene. Evidence that the inserted TH gene product was synthesizedand biologically active in vitro was provided by histological andbiochemical measures. Cells containing the TH gene showed positiveimmunoreactivity for TH in vitro. And, in the presence of the pterincofactor for TH, tyrosine present in the cell culture medium wasconverted to L-dopa through TH enzymatic activity.

The fundamental advantage of primary fibroblast cells for geneticmanipulation is the potential for using these altered cells as donormaterial for autotransplantation and thus minimizing problems withimmunological responses from the host. The primary fibroblasts used inthe present study were not implanted within the same donor animal;rather, genetically inbred Fischer 344 rats were used as both donors ofthe fibroblasts and recipients of the altered cells. This isograftapproach was selected as it simplified the maintenance of the cells invitro, while still providing conditions which approximated those ofautotransplantation. Following iso-implantation into the brain of ratswith 6-OHDA lesions, it was found that primary fibroblasts containinggenes for either βGal or TH survived for at least 10 weeks. Further,implanted fibroblasts continued to express the βGal and TH transgenesthrough the 2-monthly post-grafting survival period. Evidence that theTH-containing cells continued to produce and secrete L-dopa in vivo wasprovided by a behavioral measure: a significant decrease in rotationalasymmetry was observed only for those rats with grafts of TH-containingfibroblasts.

Fibroblast survival within the brain

When examined at 2 months, FF2/TH grafts were observed to be larger thanFF2/βGal grafts. The reason for this difference is not known.. There aremany factors which may contribute to the survival of primary fibroblastswithin the brain, including the state of the cell prior to implantationand the time that cells remain in culture prior to grafting. The FF2/THcells used in the present study were a subclone of a TH-expressingfibroblast, while the FF2/βGal cells were a bulk population ofβGal-expressing fibroblasts. The subcloning procedure which caused theFF2/TH cells to undergo more doublings than the bulk FF2/βGal cells mayhave influenced the ability of the fibroblasts to survive in vivo.Through early passages there does not appear to be a correlation betweenthe size of a fibroblast graft and the number of cell divisions inculture prior to grafting as revealed in Example IV. Fibroblasts carriedlonger in culture, as were the FF2 cells used in the present study, havebeen seen to survive well within the brain over 6 months withessentially no change in graft size or composition after 4-6 weeks.

Additional factors which may affect fibroblast survival within the braininclude the accessibility of implanted cells to an adequate nutrientsupply. Vascular processes were evident within FF2/TH grafts and lessobvious within the smaller FF2/βGal grafts. The time course for thedevelopment of a vascular supply to the grafts may also affect thelong-term survival of the fibroblasts. Grafts containing other celltypes, such as fetal neurons, have been found to be dependent on rapidvascularization for survival post-implantation (Stenevi et al., BrainRes. 114: 1-20 (1976)). Whether blood vessels within the graftsestablish a barrier to macromolecules, as typical of brain vasculature,or remain leaky, as may be typical of grafts of peripheral origin(Rosenstein and Brightman, J. Comp. Neurol 250: 339-351 (1986), may alsoinfluence the survival of implanted fibroblasts. A rupture in brainvasculature following grafting has been suggested to provide a "window"into the brain for factors which are not typically available to cellswithin the brain (Rosenstein and Brightman, supra; Sandberg et al., Exp.Neurol. 102: 149-152 (1988)). Such factors may be beneficial to cellswhich have been maintained in a serum-enriched culture medium (Gibbs etall, Brain Res. 382: 409-415 (1986); Ezerman, Brain Res. 469: 253-261(1988)). Conversely a vascular portal may place the grafted cells in aposition vulnerable to attack from circulating macrophages.

Composition of the graft

Although implanted cells were found to survive through a 2-month period,all grafts were characterized by a core of large, hemasiderin anddebris-filled cells which initially suggested a sustained macrophageinfiltration. However, while a limited number of ED-1-positivemacrophages were observed scattered around the periphery of some grafts,cells within the core of the graft were not immunoreactive with eitherED-1 or OX-42, markers which are specific for microglia andmonocytes/macrophages. Further, when grafts were stained for βGal, anenzyme which had previously been shown to exhibit a distinct granularappearance within endogenous macrophages (Shimohama et al., supra,1989), none of the large core cells within either the FF2/TH or FF2/βGalgrafts displayed granular staining. However, some of the core cellswithin the FF2/βGal grafts did display a diffuse and cytoplasmic βGalreactivity, labelling which has previously been shown to becharacteristic of immortalized fibroblasts which contain the βGaltransgene (Shimohama et al., supra, 1989). Finally, some of the largecore cells within the FF2/TH grafts displayed positive hybridization toTH mRNA. These observations strongly suggested that the phagocytic cellswithin the core of the fibroblast grafts were derived from the originaldonor cell suspension.

Mechanisms of Graft Function

The possibility that the fibroblast implantation produced an artifactual"recovery" in behavior due to damage of striatal tissue and anassociated loss of postsynaptic dopamine receptors was considered.However, there were two pieces of evidence which suggested that this wasunlikely. First, FF2/βGal rats implanted with the same volume and numberof cells as the FF2/TH rats did not show a significant change inrotational behavior following grafting. Second, destruction of intrinsicstriatal neurons and receptors with kainic acid resulted in apost-damage rotational profile which was distinctly different from thatobserved for FF2/TH rats. Rats with the smallest KA lesions, or thosewhich affected a much smaller area than that encompassed by the grafts,showed a steady decrease in rotations from 19% at 2 weeks to more than50% by 6 weeks post-damage, consistent with the known degenerativechanges which occur within the striatum following KA administration(Coyle and Schwarcz, Nature 263: 244-246 (1976)). In contrast, the mostpronounced decrease in rotations observed for FF2/TH grafted ratsoccurred 2 weeks after grafting (65%), followed by a gradual increase toa stable, but higher, number of rotations between 6-8 weeks (30% belowbaseline). This apparent decrease in graft efficacy between 2 and 8weeks may result from several different mechanisms. First, some of thegrafted cells may be lost during the initial weeks after grafting. Inother studies, fibroblast grafts have been found to shrink in sizewithin the brain through 3 weeks following implantation. After thistime, a stable graft size is attained which persists for at least 6months. A second reason for reduced graft efficacy at long post-graftingintervals may involve an alteration in the metabolic properties of thefibroblasts. Established fibroblasts may assume properties of quiescentcells with reduced metabolic activity (Dean et al., J. Biol. Chem. 261:9161-9166 (1986); Rao and Church Exp. Cell Res. 178: 449-456 (1988)),and decreased production of L-dopa. Finally, graft effectiveness may becompromised by genetic events such as deletion or rearrangement of theTH cDNA inserted in the fibroblasts, or epigenetic shut-off oftranscription from the LTR promoter driving transcription of the THcDNA. Such occurrences have been documented in other retroviral vectors(Xu et al., supra, 1989).

One final possibility which could account for a reduction in apomorphinerotations of the FF2/TH-implanted rats is that the grafts may havedisrupted cortico-striatal interactions through non-specific damage tocortical tissues. Such damage is reported to reduce rotational behaviorof 6-OHDA-lesioned rats (Freed and Canon-Spoor, Behav. Brain Res. 32:279-288 (1989)). However, in our previous work with immortalized Rat-1fibroblasts which exhibited uncontrolled growth within the brain,extensive damage restricted to cortical tissues was not associated witha drop in apomorphine-induced rotational behavior. As the graftsexamined in the present study exhibited much more limited graftextension into cortical tissue than was seen for Rat-1 grafts, it seemsunlikely that the decreased rotations of FF2/TH rats reflectednon-specific damage to cortico-striatal pathways.

Currently, L-dopa is the most effective treatment of the symptoms ofParkinson's Disease (PD). However, prolonged systemic administration ofL-dopa results in undesired side effects, such as dyskinesias and an"on-off" phenomena (Marsden and Parks, Lancet 7: 292-296 (1976)). It hasbeen suggested that some of these problems result from fluctuatinglevels of L-dopa in the plasma following oral ingestion of L-dopa, andmay be alleviated by continuous infusion of L-dopa (Nutt et al., NewEng. J. Med. 310: 483-488 (1984); Quinn et al., Neurology 34: 1131-1136(1984)). Intracerebral grafts of cells genetically modified to produceL-dopa or dopamine may provide an alternative for controlling thesymptoms of PD, and minimizing untoward side effects of extended L-dopaadministration, by providing a continuous, local infusion of dopaminedirectly to appropriate target areas within the brain.

These results demonstrate that primary fibroblasts genetically modifiedto express a transgene provide viable donor cells for intracerebralimplantation and a useful strategy for the treatment of humanneurological disease such as PD.

EXAMPLE VI Ability of Aged Fibroblasts to Serve as Donor Cells

This example demonstrates that aged human fibroblasts may be useful asdonor cells for intracerebral grafting.

Retrovital construct for Human Nerve Growth Factor ((NGF)

A molony murine leukemia virus vector (MoMLV) containing the human NGFcDNA (obtained from Syntex Research, Palo Alto, Calif.), pLhNRNL wasused. This vector was prepared by inserting the NGF cDNA into plasmidpLChRNL (see Example VII, FIG. 34) in place of the dChAT gene. The 5'long terminal repeat (LTR) drives the expression of the human NGF (hNGF)cDNA. Internal Rous sarcoma virus LTR promoter drives the expression ofthe selectable bacterial neomycin resistant gene (neo^(R))).

Production of Viral Stock

Production of infectious virus stock was accomplished by firsttransfecting the amphotropic cell line PA317 as described by Miller andButtimore, Mol. Cell. Biol. 6: 2895-2902 (1986) (provided by Dr. Miller,Fred Hutchinson Cancer Research Center, Seattle, Wash.) with the plasmidDNA by calcium phosphate co-precipitation. Conditioned media from thesecells were collected, filtered and used to infect ecotropic helper cellline psi (ψ)2 as described by Mann et al. Cell 33: 153-159 (1983). Theconditioned media from these cells were then used to infect amphotropichelper cell PA317. Producer cells from this line were selected for theirexpression of the neo^(R) gene by growing the cells in culture mediumcontaining 400 μg/ml of the neomycin analog G418 ((Gibco/BRL,Gaithersburg, Md.). Virus from the PA317 clones, producing the highesttiter (approximately 4×10⁴ c.f.u./ml) were used to infect various celllines as described below.

Infection of Cells

Primary fibroblasts, obtained from skin biopsy and established as celllines, were infected with amphotropic retrovirus. Young rat(approximately 3 months) primary fibroblasts were infected withecotropic virus expressing mouse NGF or amphotropic virus expressinghuman NGF. Primary skin fibroblasts from aged humans (male 66 years ofage with Alzheimer's and male 69 years of age without Alzheimer's) wereobtained by skin biopsy from Dr. R. Katzman, University of California,San Diego, Calif. Young (neonatal) human skin fibroblasts were obtainedfrom the Core Tissue Culture Facility, University of California, SanDiego, Calif. Rhesus monkey skin fibroblasts (10 year old male and 11year old female) were obtained by skin biopsy from the Primate Center,University of California, Davis, Calif.

Fibroblasts, grown to 70 to 80% confluency, were incubated overnightwith 10 ml of medium containing the retroviruses and 4 μg/ml ofpolybrene. The infection was performed for 3 days in a row.Subsequently, the medium was aspirated and replaced with fresh mediumcontaining 400 μg/ml of G418. After infection, the cells werecontinually maintained in G418 supplemented culture medium.

Assay of NGF Activity

NGF production and secretion by the infected fibroblasts were assayed bya two site ELISA assay using anti-β NGF antibody (Boehringer-Mannheim,Germany) as described above in Example II. NGF activity was measured inthe medium on the days indicated in FIG. 33. FIG. 33 shows the amount ofNGF secreted at different confluency from fibroblasts of differentorigin expressing either human NGF (hNGF) or mouse NGF (ratfibroblasts). In all cases the production of NGF increased or at leastremained constant (for monkey cells) during confluency. There was almostno difference in production of NGF by young or aged human fibroblastsduring log phase or confluency.

These results demonstrate that nonhuman primate (e.g. monkeys) models ofhuman disease can be used to test the efficacy of grafting geneticallymodified transgenes. In addition, these results show that aged humanfibroblasts may be successfully infected with vectors carryingtransgenes, and suggest that aged human fibroblasts may be used as donorcells.

EXAMPLE VII Regulation of the Release of Transgene Product from DonorCells

This example illustrates the regulation of secretion of a transgeneproduct, acetylcholine (ACh), from transfected cells using choline.

Cell Culture

Rat-1 fibroblasts were grown in DMEM supplemented with 0.1% glutamineand 10% FBS. Cultures were incubated in a 10% CO₂ atmosphere at 37° C.

Retroviral Construct

A Moloney murine leukemia viral vector (MoMLV) containing the DrosophilaChAT (dChAT) cDNA (obtained from Dr. P. Salvaterra, Duarte, Calif.),pLChRNL, was constructed as shown in FIG. 34. A 2519 basepair (bp)HindIII/EcoRI fragment, containing the dChAT cDNA from the plasmidpCha-2 (Itoh et al., Proc. Natl. Acad. Sci. USA 83: 4081-4085 (1986)), a377 bp EcoRI/BamHI fragment from pBR322, and a 6500 bp fragment of theretroviral construct pLLRNL (Xu et al, Virology 171: 331-341 (1989))were ligated by standard procedures (see Sambrook et al., In: MolecularCloning, A Laboratory Manual, ed. C. Nolan, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). A 5' LTR withinpLLRNL drives the expression of the dChAT gene. Similarly, an internalRSV promoter drives the expression of the selectable bacterial neomycinresistance gene (neo^(R)).

Infection

Production of infectious vector stock was accomplished by firsttransfecting the amphotropic helper cell line PA317 (Miller andButtimore, Mol. Cell. Biol. 6: 2895-2902 (1986)) with the plasmid DNA.Medium conditioned by these cells was collected, filtered and used toinfect the ecotropic helper cell line Ψ-2 (Mann et al., Cell 33: 153-159(1983)). Producer cells form this line were selected for theirexpression of neo^(R) by growing the cells in culture medium containing400 μg/ml of the neomycin analog G418 (Gibco). The two G418-resistantcolonies, from a total of 16, that expressed the highest level of ChATactivity and the highest titer of virus (approximately 10⁴ c.f.u./ml)were used to infect Rat-1 cells.

Rat-1 fibroblasts, grown to confluence on a 10 cm tissue culture plate,were split 1:5 and allowed to grow for one day before they wereincubated with 5 ml of medium containing the retroviruses and 4 μg/ml ofpolybrene. Cells were incubated overnight in this medium. Subsequently,the medium was aspirated and replaced with fresh medium containing 400μg/ml of G418. After infection, the cells were continually maintained inG418 supplemented culture medium.

Determination of ChAT Activity

The method used to determine ChAT activity is a modification of theprocedure described by Fonnum, in the J. Neurochem. 24: 407-409 (1975i,incorporated by reference herein. Fibroblasts were seeded into 35 mmtissue culture dishes and allowed to reach 70-80% confluence. Cells weresubsequently washed with PBS and collected by scraping the cells in 0.5ml homogenization buffer (0.87 mM EDTA solution containing 0.1% TritonX-100). The resuspension was transferred into a 1.5 ml Eppendorf tubeand sonicated (3X, for 10 sec). Ten microliters of the sonicatedhomogenate was placed into another Eppendorf tube. Ten microliters ofradioactive solution (0.2 mM ¹⁴ C acetyl Coenzyme A, 1 mg/ml BSA, 0.2 mMeserine salicylate, 4 mM choline chloride, 18 mM EDTA, 0.6 mM NaCl, and0.1 mM NaH₂ P₄) was added. This mixture was incubated at 37° C. for 5min. The reaction was stopped by adding 100 μl of ice cold distilledwater. Samples were extracted with 1.0 ml extraction buffer (15 g ofsodium tetraphenylboron in 850 ml toluene and 150 ml acetonitrile) andcentrifuged for 2 min in an Eppendorf microcentrifuge, after which 0.65ml of the organic phase was collected and added to 5.0 ml scintillationcocktail (National Diagnostics, Manville, N.J.). Samples were counted ina scintillation counter for 10 min. The amount of protein per culturewas determined by a Coomassie protein assay (Pierce Rockville, Ill.).

Northern Blot Analysis

Fibroblasts were seeded onto a 10 cm tissue culture plate and allowed toreach 70-80% confluence. After washing with PBS, total RNA from eachculture was isolated by extracting the cells in 0.5 ml of a guanididiumisothiocyanate solution as described by Chomczynski and Sachhi, Anal.Biochem. 162: 156-159 (1987), incorporated by reference herein. Theamount of RNA was quantified by measuring absorbance at 260 nm. Ten to15 μg of total RNA was loaded onto a 1.2% formaldehyde-agarose gel.Separated RNA was blotted onto nylon membranes (MSI) as described bySambrook et al., in "Molecular Cloning, A Laboratory Manual", (Ed.Nolan), Cold Spring Harbor Lab. Press, Cold Spring Harbor, N.Y. (1989),incorporated by reference herein. Blots were prehybridized (50%formamide, 5X Denhardt's solution, 5X SSPE, 0.5% SDS, 100 μg/mldenatured herring sperm DNA) for 1-4 hrs at 42° C. Probes were preparedby excising an approximately 2.5 Kb EcoR1 fragment from pLChRNL or a 953bp BamHI fragment from pGCyc (provided by H. Jinnah, University ofCalifornia, San Diego, Calif.) to identify dChAt or cyclophilin mRNA,respectively. The fragment was isolated on a 1.0% low-melt agarose gel,purified using Gene Clean (Bio 101, San Diego, Calif.) and labelled byrandom priming (Boerhinger Mannheim, Indianapolis, Ind.) using ³² -dCTP.Approximately 1×10⁶ cpm/10 ml hybridization buffer was used per blot.The radiolabelled probe was directly applied to the prehybridizationsolution. Hybridization was performed for 12-20 hrs at 42° C.Afterwards, the blot was washed two times with 6X SSC, 0.1% SDS atambient room temperature, two times with 1X SSC, 0.1% SDS at 42° C., andfinally once with 0.1X SSC, 0.1% SDS at 65° C. All washes lasted for 10min. The washed blot was wrapped in plastic film and autoradiographed(XAR film, Kodak, Rochester, N.Y.). The probe was removed for subsequenthybridizations by incubating the blot in 50% formamide, 10X SSPE at >65°C. for 1 hr. The blot was rinsed in 2X SSPE before the nextprehybridization. Densimetric scans of autoradiographs were conducted ona LKB ultrascan XL laser densitometer.

Immunocytochemistry

Fibroblasts were seeded at a density of 1×10⁴ cells onto two-welledtissue culture slides (Tissue Tek) and allowed to grow for two to threedays before they were fixed with 4% PBS-buffered paraformaldehyde.Cultures were permeabilized with 0.25% Triton-PBS for 15 min, washedtwice with PBS, and incubated with primary antibody at ambient roomtemperature overnight. The primary antibodies used were arabbit-anti-dChAT (1:100, from Dr. P. Salvaterra, Duarte, Calif.),mouse-anti-vimentin (1:250, Vector Laboratories), or a rabbit-anti-ratChAT (1:7500, Boehringer Mannheim). In most cases, cultures weresimultaneously incubated with antibodies against vimentin and one or theother ChAT antibody. After the primary incubation, cultures were againwashed with PBS (twice) and incubated for 1 hr at 37° C. withfluorescent labelled goat-anti-rabbit (1:100, Vector Laboratories) andgoat-anti-mouse (1:100, Chemicon, Temecular, Calif.), secondaryantibodies. Slides were mounted in Hydromount (National Diagnostics) andviewed with an Olympus fluorescent microscope.

Serum Starvation and Confluence-Induced Quiescence

Rat-1 cells were plated at a density of 2.5×10⁵ cells per 60 mm plate.For serum starvation experiments, cells were allowed to reach 70-80%confluence before the culture medium was aspirated and replaced withfresh DMEM only. Control cells were fed normal serum-supplementedmedium. Incubation lasted for 20 hr.

For confluence-induced quiescence, cells were plated as described.Cultures were then maintained for 3, 7, and 14 days after they reachedconfluence. To feed these cultures, half of the culture medium wasaspirated and replaced with an equal volume of fresh medium. The lastfeeding was three days before the cells were to be assayed. Culturescontaining actively growing cells served as controls. For Northern blotanalysis, cells were plated in 10 cm plates and treated as described.

[Methyl]-³ H-thymidine incorporation

Incorporation of thymidine was used to monitor the proliferative rate offibroblasts in logarithmic or quiescent growth state. Cells were platedonto a 10 cm culture dish and incubated with ³ [H]-thymidine (25Ci/mmol; 0.5 μCi/ml; Boehinger Mannheim) for 5 hr. Cultures weresubsequently trypsinized, counted using a hemacytometer, pelleted, andresuspended in 1 ml of 0.3M NaOH. Incubation in the NaOH solution lastedfor 30 min. The entire lysate was then pipetted into a scintillationvial containing 10 ml of scintillation fluid and counted on a Mark IIIliquid scintillation system, model 6881 (TracorAnalytic, Austin, Tex.).

Measurement of ACh by HPLC

ACh was measured by HPLC with electrochemical detection. ACh wasseparated from choline on a weak cation exchange column. This column wasprepared by loading a reverse phase Chrompher Cartridge C18 column (100mm×3 mm, Chompack) with laurylsulphate. An enzymatic post-column reactor(10×2.1 mm, Bioanalytical Systems, West Lafayette, Ind.) containingimmobilized acetylcholinesterase (type VI-S) and choline oxidase (Sigma,St. Louis, Mo.) was used to convert ACh to hydrogen peroxide. Thehydrogen peroxide was subsequently measured by electrochemical detection(Bioanalytical Systems) at a platinum electrode held at a potential of+0.5 v against a Ag/AgCl reference electrode. Mobile phase containing0.1M potassium phosphate and 0.1 mM tetramethylammoniumhydroxide at pH8.1 was delivered using a LKB 2248 isocratic pump (Pharmacia,Piscataway, N.J.) at a flow rate of 0.8 ml/min.

Medium conditioned by fibroblasts was filtered through a 0.22 μmMillipore filter and injected directly onto the analytical columnwithout any further sample preparation. Intracellular ACh levels weremeasured by resuspending the pellet, sonicating the suspension, removingcellular debris by centrifugation, and then filtering the sample firstthrough a 0.45 μm and then a 0.22 μm Millipore filter. The remainingfiltrate was then injected onto the analytical column.

Effect of Choline Chloride and Acetyl-dl-Carnitine on ACh production

Fibroblasts were plated at 1×10⁵ cells per 35 mm plate and allowed toreach 70-80% confluency. The medium was subsequently aspirated andreplaced with fresh medium containing the appropriate amount of cholinechloride (Sigma) or acetyl-dl-carnitine (Sigma). Cells were incubated inthis medium for 20 hr. Fibroblasts were harvested and prepared eitherfor the ChAT assay or for measurement of ACh by HPLC. Cultures to beanalyzed by HPLC had 0.1 mM eserine included in the medium.

Statistical Analysis

Student's t-test and a one way ANOVA statistical analysis (STATView,Palo Alto, Calif.) were used to determine differences betweenexperiments. A post-hoc Dunnet t test was used to determine individualdifferences between groups.

Confirmation of dChAT Expression by Rat-1/dChAT Fibroblasts

Rat-1 Fibroblasts were infected with retrovirus (FIG. 34) and selectedin G418-containing medium. The presence of dChAT in infected cells wasinitially confirmed by immunocytochemistry. Uninfected Rat-1 (control)and Rat-1/dChAT fibroblasts were simultaneously stained with antibodiesagainst vimentin and dChAT or vimentin and rat-ChAT (FIG. 35a-f).Vimentin immunoreactivity effectively revealed the morphology of thefibroblasts (FIG. 35a-c). Most Rat-1/dChAT cells were morphologicallyindistinguishable from controls, displaying a flat, epithelial-likephenotype. Some lines of Rat-1/dChAT fibroblasts were observed to bemorphologically distinct from controls, exhibiting an elongatedmorphology.

As expected, anti-dChAT antibodies labeled only Rat-1/dChAT fibroblasts(compare with FIG. 35d and 35e). Staining of dChAT was evenlydistributed throughout the cytoplasm; however, there was an area of moreintense immunoreactivity immediately around the nucleus. The nucleusitself was not stained. In addition to staining for the presence ofrecombinant dChAT, Rat-1 and Rat-1/dChAT cells were also stained for thepresence of rat-ChAT. There was no indication of rat-ChAT immunostainingin either control or Rat-1/dChAT cells (FIG. 35f). The lack of rat-ChATimmunoreactivity indicates that there is very little, if any, endogenousChAT activity in Rat-1 cells. Moreover, these observations demonstratethat the dChAT molecule is immunologically distinct from rat-ChAT.Previous biochemical and molecular studies have reported sequencedifferences between dChAT and the other mammalian ChATs that have beencloned (Berrard et al., Proc. Natl. Acad. Sci. USA 84: 9280-9284 (1987);Itoh et al., Proc. Natl. Acad. Sci. USA 83: 4081-4085 (1987)). Thesedata show that dChAT, in addition to catalyzing the formation of ACh,may be used as a specific marker for dChAT-expressing fibroblasts thathave been transplanted into the rat CNS.

Total RNA from Rat-1 and Rat-1/dChAT fibroblasts was also compared byNorthern blot analysis to determine specificity of expression of thetransgene. The results from these studies clearly demonstrate that themRNA encoding for dChAT is present only in Rat-1/dChAT fibroblasts (FIG.36a). The size of the dChAT mRNA isolated from Rat-1/dChAT cells wasapproximately 6 kb. The size of the mRNA is due to the lack of apolyadenylation sequence between the dChAT cistron and RSV promoter.This allows unimpeded transcription to proceed from the 5' LTR to the 3'LTR.

Finally, control and Rat-1/dChAT fibroblasts were assayed for ChATactivity. The level of ChAT detected in Rat-1 cells was widely variable,ranging from no activity to approximately 6.4 nmoles ACh/hr/mg protein.The average activity displayed by control cells was approximately1.9±1.1 nmoles ACh/hr/mg protein (FIG. 36b). In contrast to the lowlevels of ChAT detected in Rat-1 cells, ChAT activity in Rat-1/dChATcells was >1200-fold higher. The average activity of Rat-1/dChATcultures was 2397±149 nmoles ACh/hr/mg protein (FIG. 36b). Moreover,expression of dChAT by transduced fibroblasts was stable. The activityof ChAT in fibroblasts that were continuously passaged for one yearafter infection was similar to that in fibroblasts assayed one weekafter infection.

Rat-1/dChAT Cells Produce and Secrete ACh

The demonstration that transduced Rat-1 cells express an activerecombinant enzyme capable of acetylating choline in an in vitro assaydoes not prove that the cells are capable of manufacturing and extrudingACh from the cytoplasm. Therefore, the level of ACh found in the culturemedium and within the cells was analyzed by HPLC with electrochemicaldetection. No ACh was found either within the cells or in the nutrientmedium of uninfected Rat-1 cells (FIG. 36c). This result suggests thatthe ChAT activity detected in Rat-1 cells is due to a contaminatingenzyme which is capable of acetylating choline. Carnitineacetyltransferase, an enzyme found in most cells, is capable oftransferring the acetyl moiety from acetyl-CoA to choline (White and Wu,Biochemistry 12: 841-846 (1973)). In contrast to the absence of ACh incontrol cultures, significant levels of ACh, both intra- andextracellular, were measured in Rat-i/ChAT cultures (FIG. 36c). Theaverage cellular content of ACh was approximately 0.78±0.23 nmol.Comparatively, approximately 1.7±0.10 nmol of ACh was found in theculture medium.

These results demonstrate that ACh is actively being manufactured byRat-1/dChAT fibroblasts and that most of the ACh is released from thecells into the surrounding culture medium.

Effect of Choline Chloride and Acetyl-dl-Carnitine on ChAT and AChExpression

Because choline chloride and acetylcarnitine have been reported toenhance ACh secretion from neurons or synaptosomes in various modelsystems (Gilson and Shimada, Biochem. Pharmacol. 29: 167-174 (1980);Imperato, Neurosci. Lett. 107: 251-255 (1989); Jenden et al., Science19: 635-637 (1976); Tucek, J. Neurochem. 44: 1-24 (1985)), these drugswere tested for their ability to modulate ACh secretion and ChATactivity in Rat-1/dChAT fibroblasts. Choline chloride was used inconcentrations ranging from 0.25-500 μM (FIG. 37a). These concentrationswere in addition to that normally found in the medium (approximately 28μM) and in the serum (<2 μM). Exogenous choline concentrations of0.25-5.0 μM had no affect on ACh levels. At 10 μM, both intracellularand secreted ACh concentrations increased (FIG. 37a). Intracellularlevels of choline increased by approximately 2-fold; the concentrationof ACh found in the culture medium increased by approximately 1.8-fold.Intracellular levels of ACh continued to rise with increasingconcentrations of choline (FIG. 37a). The addition of choline in amountsgreater than 500 μM did not cause any further increase in ACh content.

In contrast to the consistent rise in intracellular ACh, secreted AChdeclined back to baseline levels at 50 and 100 μM choline afterincreasing by 80% at 10 μM choline (FIG. 37a). At 200 μM choline,released levels of ACh increased by approximately 2.0-fold. AChcontinued to increase at 500 μM choline, reaching 2.6 times the levelsobserved in control (no added choline) cultures. Concentrations above500 μM choline did not elicit any further increase in ACh release. Theaddition of choline (up to 10 mM) to the medium of uninfected Rat-1cells did not result in the detection of ACh as determined by HPLC.

The effect of choline on ChAT activity was also determined (FIG. 37b).Concentrations of 10, 200 and 500 μM choline were tested. The additionof choline to the culture medium did not significantly affect the ChATactivity of Rat-1/dChAT fibroblasts.

The influence of acetyl-dl-carnitine on ACh was also tested.Acetyl-dl-carnitine had no effect on the amount of ACh produced orreleased from Rat-1/dChAT fibroblasts or on ChAT activity.

Effect of Serum Starvation and Confluency on the Expression of dChAT

Most of the characterization of Rat-1/dChAT cells has been conducted oncultures of proliferating fibroblasts. However, the ultimate goal is touse fibroblasts for grafting. Fibroblasts that have been transplantedinto the brain are most likely not actively proliferating. Thus, the invitro activity of quiescent fibroblasts, which may more accuratelyreflect the conditions found in vivo, should be assessed. Thisassessment provides predictive information about the activity ofnon-dividing fibroblasts that have been implanted into the brain. Toinduce quiescence in fibroblast cultures, Rat-1/dChAT fibroblasts weremaintained in either a serum-free medium or at high density for severaldays. Although both methods significantly reduced thymidineincorporation, maintaining fibroblasts in a post-confluent state for aprolonged period resulted in much lower thymidine incorporation (Table3).

                  TABLE 3                                                         ______________________________________                                        [.sup.3 H]-Thymidine Incorporation                                            Culture Condition                                                                          CPM/10.sup.5 Cells                                                                        Cells/ml  % Control                                  ______________________________________                                        Log, 10% serum                                                                              3.6 × 10.sup.4                                                                     9.9 × 10.sup.5                                                                    --                                         (control)                                                                     No Serum      3.7 × 10.sup.3 *                                                                   4.7 × 10.sup.5                                                                    10.3                                       14 DPC, 10% Serum                                                                          55.4 ± 8.5*                                                                            3.0 × 10.sup.6                                                                    <0.2                                       ______________________________________                                         *Significance of p < 0.01                                                     14 DPC = 14 days post confluence                                         

FIG. 38a illustrates the effect of serum starvation on ChAT activity.This treatment did not noticeably affect the morphology or the health ofthe fibroblasts. In contrast, maintaining Rat-1/dChAT fibroblasts inserum-deficient medium caused a marked reduction in the expression ofthe transgene. The absence of serum caused an approximately 80% decreasein ChAT activity.

Northern blot analysis was conducted to determine if the steady statelevel of dChAT mRNA decreased with serum deprivation. There was asignificant decrease (approximately 46%) in the level of dChAT mRNA whenserum was withdrawn from the cultures (FIG. 38b). The same blots werealso probed with a cDNA probe to cyclophilin which served as an internalcontrol (McKinnon et al., Mol. Cell. Biol. 7: 2148-2154 (1987)).Unexpectedly, the steady state levels of cyclophilin mRNA also appearedto decrease (approximately 43%) in serum-starved fibroblasts. Theabsorbance ratio of dChAT to cyclophilin was measured with adensitometer to confirm that the observed decreases in the steady statelevels of dChAT and cyclophilin mRNA were proportional. Indeed, theratio of the relative absorbance of dChAT to cyclophilin was similar incontrol and serum-starved cultures (FIG. 38c). The amount of actin andHPRT mRNA was also reduced in serum-starved fibroblasts.

Since serum deprivation appeared to affect the general metabolism offibroblasts, an alternative method was employed in which quiescence wasinduced by continuing to maintain fibroblasts for 3, 7 and 14 days afterthey reached confluency (days post confluence, DPC). At 3 DPC, the levelof ChAT declined to 80% of the activity observed in control, growingcultures (FIG. 39a). The activity of ChAT continued to drop withincreasing DPC, decreasing to 50% and 20% of control levels at 7 and 14DPC, respectively (FIG. 39a). Maintaining the cells beyond 14 DPC didnot result in a further decline in ChAT activity.

The amount of dChAT mRNA in confluent Rat-1/dChAT cultures wasdetermined by Northern blot analysis (FIG. 39b). There was no obviousdecrease in the level of dChAT mRNA isolated from growing (control) orconfluent Rat-1 cultures, however, densitometric analysis revealed thatthe steady state level of dChAT mRNA decreased by approximately 29% andapproximately 35% at 3 and 14 DPC, respectively. Interestingly, as therelative levels of dChAT mRNA decreased, the levels of cyclophilin mRNAincreased. As compared to controls, the relative amount of cyclophilinmRNA isolated from 3 and 14 DPC cultures increased by approximately 84%.Determination of the dChAT to cyclophilin ratio indicated that a 60.5%decrease occurred at 3 DPC and a 64% decrease occurred at 14 DPC (FIG.39c). All numbers are relative to the dChAT/cyclophilin ratio determinedfor growing cells.

Effect of Choline on the release of ACh from Confluent Fibroblasts

To assess whether choline could enhance the release of ACh fromconfluent fibroblasts, Rat-1/dChAT cells were maintained for 7 DPC ineither control or choline-supplemented (500 μM) medium. FIG. 40illustrates the effects of maintaining Rat-1/dChAT fibroblasts in aconfluent state on ACh production and release. The overall content ofACh (intra- and extracellular) in 7 DPC Rat-1/dChAT cultures was 18.9%of that measured in cultures containing mitotic cells. The decrease intotal ACh was manifested as a 50.1% and 82.1% decline in intracellularand released ACh, respectively. In contrast to confluent culturesmaintained in normal medium, quiescent Rat-1/dChAT fibroblastsmaintained in choline-supplemented medium demonstrated ACh levels thatwere comparable to the levels of ACh measured in proliferatingRat-1/dChAT cultures (FIG. 40). Intracellular ACh levels increased by138%, as compared to confluent fibroblasts maintained in normal medium,when exogenous choline was added to the nutrient medium. Similarly, theamount of extracellular ACh increased by 473%.

The above results set forth in this example demonstrate that Rat-1fibroblasts can be successfully infected with a retroviral vectorcontaining the cDNA encoding dChAT. Moreover, these cells canmanufacture and, more importantly, release the product of ChATcatalysis, ACh. The production and secretion of ACh from proliferatingfibroblasts can be modulated if additional choline chloride is added tothe culture medium. These data also show that the expression of thetransgene is decreased in quiescent fibroblasts; however, choline canenhance the release of ACh from these cells.

Rat-1/dChAT cells were developed so that some of the factors which mayaffect ACh production could be studied in vitro. Rat-1 fibroblasts werechosen because they can be continuously maintained in culture withlittle, if any, change in their genotype or phenotype. The stability inthe expression of the transgene in these cells is reflected by theobservation that ChAT activity was similar in cells assayed one week orone year after retroviral infection. A short-term goal for developingACh-secreting fibroblasts is to assess the function of these cellsfollowing implantation into the CNS. However, Rat-1 and other fibroblastcell lines implanted into the rat CNS often form tumors (see Horellou etal., Neuron 5: 393-402 (1990); Wolff et al., Proc. Natl. Acad. Sci. USA86: 9011-9014 (1989)). By contrast, primary dermal fibroblasts cansurvive for extended periods in the brain (6 mos.) without anyindication of tumor development (see Fisher et al., Neuron 6: 371-380(1991); Kajawa et al., J. Comp. Neurol. 308: 2-13 (1991)). Thus, ifACh-secreting fibroblasts are to be used in brain transplant paradigms,primary cells should be used. Primary dermal fibroblasts appear to reactin a similar manner to Rat-1/dChAT cells with regards to choline andquiescence (i.e., increased ACh secretion and decreased ChAT activity,respectively). Therefore, the Rat-1 fibroblasts used in this Exampleserve as an accurate in vitro model for the reaction of primaryfibroblasts grown under similar culture conditions.

To enhance the ability of cells transplanted into the CNS to produce afunctional effect, it would be useful if the secretion of the moleculeof interest could be modulated by manipulating the humoral environmentwithin the CNS. In the in vitro experiments described herein, cholineincreased the production and secretion of ACh by Rat-1/dChATfibroblasts. Importantly, choline also elicited an increase in AChproduction and release from quiescent (confluent) fibroblasts. Thislatter observation may provide a means to modulate ACh production byChAT-producing cells transplanted into the CNS. At present, themechanism behind the increase in intra- and extracellular ACh caused byexogenous choline is not known. However, the amount of enzyme, asassessed by measuring ChAT activity, is not affected by increasing theextracellular concentration of choline. These results indicate that therate of the forward reaction, i.e. towards the formation of ACh and notthe amount of enzyme, is responsible for the increased level of AChobserved in choline-supplemented fibroblast cultures.

These results suggest that the release of ACh may be increased indChAT-expressing fibroblasts that have been implanted into the brain byadministering choline, for example as a dietary supplement. Choline hasbeen reported to pass through the blood-brain barrier (Cohen andWurtman, Life Sci. 16: 1095-1102 (1975); Cohen and Wurtman, Science 191:561-562 (1976); Wecker and Schmidt, Brain Res. 184: 234-238 (1980)).

In addition to the possible modulation of the secretion of transgenicproducts, another desirable quality of genetically modified cells isstable, long term expression of the recombinant gene after the modifiedcells have been transplanted. Fibroblasts implanted into the brainsurround themselves with copious amounts of collagen and apparentlycease to proliferate (Kawaja et al., supra, 1991). Current work stronglysuggests that the expression of transgenes from the MoMLV LTR isdramatically decreased in serum starved and contact-inhibited quiescentfibroblasts. The mechanisms of action behind serum-starved andconfluence-induced quiescence are not clear, but they do appear to bedifferent. This is evidenced by the observation that dChAT mRNA and ChATactivity decreases more rapidly in serum starved fibroblasts than incultures containing contact inhibited cells.

Current data suggests that once primary fibroblasts are implanted intothe brain, the expression from a proviral transgene in these cells isreduced. This hypothesis is supported by previous studies that haveshown that TH-expressing 208F fibroblasts, which can be immunolabeledfor TH in vitro, are not effectively stained once implanted into thebrain (Wolff et al., supra, 1989). Primary fibroblasts that express thegene for GAD also demonstrate a marked decrease in GAD activity whenthey are transplanted into the brain (Chen et al., J. Cell. Biochem.,45-252-257 (1991)). More recent in situ hybridization andimmunocytochemical studies have indicated that a residual amount of THmRNA and protein are present in late post-transplantation (ten weeks)primary fibroblasts (Fisher et al., supra, 1991)). These results,coupled with those presented in this example, demonstrate that althoughthe amount of recombinant product decreases in quiescent fibroblasts,there is a residual amount still present in the cells. Results show thata baseline level of dChAT activity (approximately 20% of that found inproliferating cells) exists in quiescent Rat-1/dChAT fibroblasts. Thisamount of activity does not decrease by maintaining the fibroblasts in aconfluent state for periods longer than 14 DPC. Thus, if the activity ofthe transgene is high enough (as measured in confluent cells in vitro),the amount of activity remaining once the cells have been transplantedmay be enough to elicit a functional effect. This hypothesis issupported by the observation that increased amounts of substrate(choline) enhance the release of ACh from quiescent Rat-1/dChATfibroblasts. Therefore, the drop in the expression of some transgenesmay be compensated for by defined epigenetic factors.

In conclusion, these results demonstrate that fibroblasts can begenetically altered to produce and secrete ACh. ACh release can beincreased by manipulating the extracellular concentration of choline,possibly by administering dietary choline. These data also demonstratethat transgene expression markedly decreased with the cessation ofproliferation in vitro.

EXAMPLE VIII Use of Promoters In Quiescent Fibroblasts

This example provides a comparison of promoters in growing (Log) andconfluent (quiescent) fibroblast cultures. The reporter transgene usedin this study was chloramphenicol acetyltransferase (CAT).

Expression Vectors

Expression vectors containing the CAT gene under the control of variouspromoters for determining transgene expression in proliferating andquiescent primary fibroblasts, were obtained. The plasmids, pSV2CAT(Gorman et al., Mol. Cell. Biol. 2: 1044-1051 (1982); Subramani andSouthern, Anal. Biochem. 135: 1 (1983); ATCC No. 37155) in which the CATgene is under the control of the SV40 promoter plus the SV40 enhancersequence, and pSV232ACAT (FIG. 41, provided by Dr. D. Jolly and Dr.Theodore Friedmann, University of California, San Diego, Calif.,constructed from pSV232Agpt described by Fromm and Berg, J. Mol. Appl.Genet. 2: 127-135 (1983)), were used. Plasmids pRSVCAT (Rous sarcomavirus long terminal repeat promoter and enhancer, Gorman et al., Proc.Natl. Acad. Sci. USA 79: 6777-6781 (1982); ATCC No. 37152), pMLVCAT(FIG. 42, murine Moloney leukemia virus long terminal repeat promoterand enhancer, constructed from pSV232ACAT, pGEM-3 (Promega Corp.,Madison, Wis.) and pN2 (Eglitis et al., Science 230: 1345-1398 (1985)and provided by Dr. M. Rosenberg, University of California, San Diego,Calif.) and pCMVCAT (FIG. 43, human cytomegalovirus immediate earlypromoter and enhancer, constructed by Dr. M. Rosenberg from pSV232ACATand pON249 (from E. S. Mocarski, Stanford University School of Medicine,Palo Alto, Calif.)) were provided by Dr. Friedmann (University ofCalifornia, San Diego, Calif.). Collagen promoters were supplied by Dr.de Crombrugghe, M. D. Anderson Cancer Center, Houston, Tex. PlasmidspG100 and pAZ1003 (de Crombrugghe and Schmidt, Methods in Enzymology266: 61-76 (1987)), were provided by Dr. de Crombrugghe (Houston, Tex.)and contain the CAT gene under the control of mouse α1(I) and α2(I)collagen promoters (de Crombrugghe, supra, α2(I); Thompson et al.,Annals New York Acad. Sci. 580: 454-458 (1990)) α1(I)).

Because CAT is a bacterial gene with no mammalian equivalent, any CATactivity detected in transfected cells is due to the presence of thetransfected plasmid. The level of CAT enzyme in transfected cells isproportional to the strength of the promoters (Gorman et al., Proc.Natl. Acad. Sci. USA 79: 6772 (1982)).

Establishment of Fibroblast Cultures

To obtain rat primary skin fibroblasts, the abdomens of twenty young(200-250g) anesthetized Fischer 344 rats were soaked with 70% ethanol.25×25 mm biopsies were removed from the abdomen of each rat andimmediately dipped in 70% alcohol for 2-3 min. The tissue was thentransferred to phosphate-buffered saline (PBS, pH 7.4) to remove thealcohol. The biopsy material was then placed into a solution containing10 mg/ml of collagenase in PBS and incubated for 15 min. at 37° C. Thedermal layer was removed from the epidermal layer with the aid of finetweezers and subject to further enzyme digestion for 0.5 hr. The cellswere then pelleted and plated into 35 mm² tissue culture dishes andmaintained in Dulbecco's minimal essential medium (DMEM, Gibco, GrandIsland, N.Y.) supplemented with 10% fetal bovine serum (FBS). Allcultures were maintained in a 10% CO₂ atmosphere at 37° C. and werepassaged at a split ratio of 1:2 upon reaching >80% to 90% confluency.Transfected fibroblasts were grown to 70% to 80% confluency for assay ofCAT activity during log phase. For assay of CAT activity in confluency,cells were grown to 100% confluency and then maintained for anadditional 7 days before assay.

Transfection of Fibroblasts

Fibroblasts were transfected with the selected plasmid in a ratio of10:1 plasmid DNA to pRSVneo^(R) plasmid using Lipofectin reagent (BRL,Inc., Gaithersburg, Md.) as described by Felgner et al., Proc. Natl.Acad. Sci. USA 84: 7413-7417 (1987) and Felgner and Holm, Focus 11:21-25 (1988), both incorporated by reference herein. For each 60 mm dish(5×10⁵ cells/dish) different amounts of DNA and 30 μg Lipofectinreagent/3 ml Opti-MEM media (BRL, Inc.) were used.

For selection of stable transformants plasmid DNA containing the neo^(R)gene under the control of RSV/LTR was mixed with vector DNA containingthe reporter gene (CAT) under the control of various promoters at aratio of 1:10. After washing once with Opti-MEM, fibroblasts wereincubated in the presence of the DNA/Lipofectin mixture for 5 h at 37°C. After that time, the media were changed with DMEM, 10% FCS andincubated for 24-36 h before selection.

The stable transformants were selected in the presence of 200 μg/mlG418. The transfected cells were passaged when the plate was at least50% confluent. Transfected fibroblasts were split at a 1:2 ratio uponreaching >80% confluency.

Transfected cells expressing CAT were assayed as described below at bothlog phase and after confluency. Results were compared to determinewhether the expression of the CAT transgene remained stable, decreasedor increased after the cells became confluent.

Assay for CAT Activity

CAT activity was assayed using a modification of the method of Sleigh,Anal. Biochem. 162: 156-159 (1987), incorporated by reference herein.The dish containing the transfected fibroblasts was washed in PBS andthe cells were collected by scraping. The cells were resuspended in0.25M Tris-HCl, pH 7.8 buffer (100 μl/plate), lysed by sonication andcentrifuged to remove the cell debris. The protein concentration of thesoluble fraction was determined using Coomassie protein reagent (PierceChemical Co., Rockford, Ill.). An equal amount of protein from eachtransfection was used for assay. In addition to cell extracts, 100 μl ofreaction mixture contained 20 μl of 8 mM chloramphenicol and 5 μl of 0.5mM cold and [¹⁴ C] acetyl-CoA. After incubation for 1 h at 37° C., thechloramphenicol and its acetylated form was extracted with 0.12 ml ethylacetate. About 80 μl of the organic phase was collected aftercentrifugation (10,000 X g) at room temperature. The reaction mixturewas then re-extracted with ethyl acetate. About 100 μl of organic phasewas collected and mixed with 80 μl of the organic phase collectedearlier. After adding 1 ml of Packard Instagel (Packard, Downers Grove,Ill.) to the organic phase the samples were counted in a scintillationcounter.

Northern Blot Analysis

To determine whether the variable expressions of the transgene duringlog or confluent stages of fibroblast growth occur at thetranscriptional level, Northern blot analysis was performed toquantitate the amount of RNA synthesized as follows. Cells grown in a 10cm plate were processed for isolation of total RNA by Northern blotanalysis at either 70-80% confluence or after maintenance in a confluentstate for 7 days. Total RNA from each culture was isolated by extractingthe cells in 0.5 ml of a guanididium isothiocyanate solution asdescribed by Chomczynski and Sacchi, in the Anal. Biochem. 162: 156-159(1987), incorporated by reference herein. Protein and DNA were separatedfrom DNA by extracting with phenol-chloroform. The amount of total RNAwas quantified by measuring absorbance at 260 nm. Ten to 15 μg of totalRNA was subsequently loaded onto a 1.2% formaldehyde-agarose gel.Separated RNA was blotted onto a nylon membrane (Hybridization TransferMembrane, MSI, Inc.) by capillary diffusion using 10 X SSC.Prehybridization (50% formamide, 5X Denhardt's solution, 5X SSPE, 0.5%SDS, 100 μg/ml denatured herring sperm DNA) of the blot was performed at42° C. for 1-2 hr.

For preparation of probes, dChAT cDNA was excised from pCMVCAT usingpstI enzymes from pLChRNL and was purified from low melting pointagarose gel as described by Maniatis et al., in Molecular Cloning-ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1982), incorporated by reference herein. ³² p labelled probe wasgenerated by random priming (Maniatis et al., supra). Denaturedradiolabeled probe (boiled for 2 min and cooled on ice) was directlyadded to the prehybridization solution. Hybridization was conducted at42° C. for 12-24 hr. The blot was then washed twice with 6X SSC, 0.1%SDS at ambient room temperature, twice with 1 X SSC, 0.1% SDS at 42° C.,and once with 0.1 X SSC, 0.1% SDS at 65° C. All washes were for 10 min.The washed gel was then wrapped in plastic XAR film and autoradiographed(Kodak, Rochester, N.Y.). The intensity of each RNA band was measured bydensitometer scanning in LKB Ultrascan XL (LKB, Sweden). Blots werewashed to remove all counts and reprobed with a cyclophilin probe foruse as an internal standard. The probe was removed for subsequenthybridizations by washing the blot in 50% formamide, 6 X SSPE at >65° C.for >30 min. The blot was rinsed in 2 X SSPE before successiveprehybridizations.

In this example, the SV40 and LTR promoters and the α2(I) collagenpromoter, both with and without the collagen enhancer were tested in logand quiescent fibroblasts. The results are summarized in FIG. 44.

The level of CAT activity in fibroblasts containing the SV40 promoter(SV40) was approximately 3247 and approximately 2712 DPM/μg protein forgrowing and quiescent cells, respectively. This is a decline ofapproximately 17%. Similarly, CAT activity in fibroblasts containing theLTR decreased by approximately 24% at confluence (3291 DPM/μg proteinfor log and 2494 DPM/μg protein for quiescent cells). In contrast, thelevels of CAT measured in fibroblasts containing the α2(I) collagenpromoter increased with quiescence, although the overall expression fromthese cells was lower. CAT activity in fibroblasts containing theenhancerless α2(I) collagen promoter (Coll) was approximately 13 DPM/μgand approximately. 75 DPM/μg protein for log and quiescent cells,respectively. This is an increase of approximately 576%. Fibroblastscontaining the α2(I) collagen promoter with the collagen enhancer(Coil(E); described by Rossi and de Crombrugghe, Proc. Natl. Acad. Sci.(USA) 84: 5590-5594 (1981)) exhibited CAT levels of 405 DPM/μg proteinfor log and 606 DPM/μg protein for quiescent fibroblasts. This is anincrease of approximately 149%.

These results indicate that transgene expression from viral promotersmay decline in quiescent fibroblasts. In contrast, transgene expressionfrom the α2(I) collagen promoter increases with quiescence. Becausefibroblasts grafted into the brain are nondividing, one way to maximizestable transgene expression is by using a promoter that is normallyactive in quiescent fibroblasts. The α2(I) collagen promoter is such apromoter. Moreover, the Coll(E) promoter-enhancer is as efficient as theα2(I) promoter in driving transgene expression under appropriate cultureconditions.

These results demonstrate a method for increasing transgene expressionin cells that have ceased dividing when implanted into the body topromote long term stable expression of therapeutic transgenes.

EXAMPLE IX Regulation of Promoters by Cytokines and Anti-inflammatoryAgents

Many mononuclear phagocytes and lymphocytes invade the CNS after theblood-brain barrier is compromised; for example after the implantationof cells into the brain. These cells are known to secrete cytokines intothe surrounding extracellular environment. The infiltration andaccumulation of these cells around grafted material is observed within aweek after grafting. Because fibroblasts have been shown to respond to avariety of cytokines this example was performed to assess the role ofcytokines in regulating LTR-driven transgene expression in primary ratfibroblasts.

This Example describes the use of 1) an anti-inflammatory agent,dexamethasone, to eliminate the effects of cytokines on the steady statelevels of LTR-driven proviral mRNA; and 2) the use of the α2(I) collagen(Coll) promoter with the collagen enhancer (Coil(E)) to take advantageof the high levels of cytokines present in the brain after implantation.The transgenes used in this example were Drosophila cholineacetyltransferase (dChAT) and chloramphenicol acetyltransferase (CAT).

Methods

The methods used in this Example were as described above for exampleVIII with the following exceptions:

Lipofection

The method of lipofection was as described above in Example VIII. Theplasmids used to transfect fibroblasts in this example were pMLVCAT (LTRpromoter supplied by Dr. Friedmann, University of California, San Diego,Calif.) and pR40 (Coll(E) promoter and enhancer (described in Rossi andde Crombrugghe, Proc. Natl. Acad. Sci. USA 84: 5590-5594 (1987), andsupplied by Dr. de Crombrugghe, Md. Anderson Cancer Center, Houston,Tex.). The dChAT-fibroblasts were transduced by infection withretroviruses as described above for Example VII.

Fibroblast cultures

Primary skin fibroblast cultures were established from rat skin asdescribed above in Example VIII with the following differences.dChAT-fibroblasts were grown to confluence in a 35 mm tissue culturedish using DMEM supplemented with 10% FBS and 200 μg/ml of G418. Uponreaching confluence, the cells were switched to medium containing 2%serum and maintained for an additional 7 days. Fibroblasts that werelipofected with the pMLVCAT or pR40 plasmids were grown to confluence asdescribed for the dChAT-fibroblasts. However, after reaching confluence,these cells were maintained in 10% serum for 4 days. They weresubsequently switched to 2% serum-containing medium for the final 3 days(for a total of 7 days post-confluency).

Treatment of Transfected Fibroblasts with Cytokines or Anti-InflammatoryAgent

Cytokines were added on the 7th day of post-confluency. TGFβ1 (10ng/ml), IL-1β (30 μ/ml), and TNFα (150 ng/ml) were incubated with thecultures for 24 hrs. In addition, the anti-inflammatory agentdexamethasone (25 μM) was added to fibroblasts alone and in the presenceof TGFβ and IL-1β1. The cells were then taken for Northern blot analysis(dChAT-fibroblasts) or CAT assay (cells containing the LTR or Coll(E)promoters). Infγ was incubated as described for the other cytokines;however, for some experiments the medium containing Infγ was aspiratedand replaced with fresh 2% serum-containing medium. The cells weremaintained in this way for an additional 48 hours before they wereprocessed for Northern blot or CAT assay. This procedure was usedbecause it was determined that Infγ required this amount of time tofunction.

Confluent, dChAT-expressing fibroblasts were incubated with TGFβ1, TNFα,IL-1β or Infγ as described. Total mRNA from these cultures was isolatedand probed with a cDNA fragment that specifically recognizes proviralmRNA for the Northern blot. FIG. 45a demonstrates that both TGFβ1 (lane2) and IL-1β (lane 3) significantly decreased the steady state level ofproviral mRNA. TNFα (lane 4), on the other hand, did not significantlyaffect the levels of proviral mRNA. FIG. 45b illustrates the effects ofInfγ on dChAT mRNA content. After 24 hrs there was no change in thesteady state level of proviral mRNA; however, mRNA levels were markedlyreduced 48 hrs after Infγ was removed and replaced with fresh medium.

These results demonstrate that cytokines affect LTR-driven dChATexpression in vitro.

FIG. 46 is a Northern blot illustrating that the co-administration ofTGFβ1 and IL-1β to confluent LTR-driven dChAT-producing fibroblastssignificantly decreased the amount of proviral mRNA contained within thefibroblasts. In contrast, dexamethasone increased the amount of dChATmRNA detected by Northern blot analysis. If dChAT-fibroblasts weresimultaneously exposed to dexamethasone, TGFβ1, and IL-1β for 24 hrs,the level of proviral mRNA was similar to that detected in controlcultures. These results suggest that anti-inflammatory agents, includingsteroids such as dexamethasone, can mitigate the combined negativeeffects of TGFβ1 and IL-1β on the steady state level of proviral mRNA invitro.

As described above, several cytokines downregulate the expression oftransgenes from the LTR. One way to minimize the effects of thesecytokines after grafting fibroblasts into the brain would be to reducethe inflammatory response using an anti-inflammatory agent such asdexamethasone. An alternative approach that would not require theadministration of anti-inflammatory drugs is the use of a promoter orpromoters that are not affected, or are possibly enhanced, by thepresence of cytokines. One such promoter is the α2(I) collagen promoter.In these experiments, the CAT gene was used as the reporter.

FIG. 47 depicts the effects of TGFβ1, TNFα, IL-1β and Infγ on theexpression of CAT in cultures of quiescent fibroblasts in which theColl(E) promoter-enhancer was used to drive transgene expression(Coll(E)-fibroblasts). For these experiments the cultures weremaintained in 2% serum containing medium for the final three days of aseven day post-confluent culture state. Surprisingly, the activity ofCAT in Coll(E)-fibroblast cultures maintained in 2% serum was 440%greater than comparable cultures maintained in 10% serum-containingmedium (FIG. 47a). This activity approached that measured for LTR(compare with FIGS. 44 (Example VIII) and 47a).

Of the cytokines tested, TGFβ and IL-1β did not appear to significantlyaffect CAT activity (FIG. 47b). Exposure to TNFα, on the other hand,resulted in an approximately 32% increase in CAT activity. In contrast,Coll(E)-fibroblasts incubated with INF₇ displayed an approximately 53%decrease in CAT activity. This measurement was made 48 hrs after Infγwas removed and replaced with fresh 2% serum-containing medium.

In vivo Expression Using Coll(E)

LTR-CAT cells (fibroblasts in which the CAT gene is driven by the LTR)and Coll(E)-CAT cells (fibroblasts in which the CAT gene is driven bythe α2(I) collagen promoter-collagen enhancer) were transplanted intothe striatum of adult Fischer 344 rats as described in Example VIIIabove. Three (3) μl of LTR-CAT or Coll(E)-CAT cells (200,000 cells/μl; atotal of 600,000 cells) was injected. LTR-CAT fibroblasts were injectedinto the right side, Coll(E)-CAT cells were injected into the left. At 1and 4 weeks after implantation, the rats were perfused, the brainssectioned, and processed for CAT immunohistochemistry. The antibody,anti-chloramphenicol, was used at a dilution of 1:2000 and was purchasedfrom 5 prime-3-prime, Boulder, Colo.

As shown in FIG. 48, many strongly stained CAT-positive fibroblasts wereobserved in grafts containing either LTR- or Coll(E)-CAT fibroblasts at1 week post-transplantation (FIG. 48a, FIG. 48b, white arrows). Theintensity of staining was comparable between the two graft types. At 4weeks, (FIG. 48c and 48d, white arrows), the staining for CAT was morerobust in Coll(E)-CAT fibroblasts grafts. There were still manyphagocytic cells associated with all grafts at this time point(Arrowheads indicate the graft-host brain interface).

These results suggest that the Coll(E) promoter-enhancer may providemore stable and stronger expression of transgene after donor cellimplantation into the brain. The presence of phagocytic cells within thecenter of the grafts at 4 weeks indicates that cytokines may still bepresent at high levels. Thus, one reason for the stronger expressionfrom the Coll(E) promoter-enhancer as compared to the LTR may be thatthe cytokines are acting to increase expression.

The data obtained in this example demonstrate that 1) some cytokines(for example, TGFβ1, IL-1β and Infγ) negatively affect the level ofproviral mRNA, and that 2) dexamethasone can counteract the negativeeffect of TGFβ and IL-1β on the steady state level of proviral mRNA andthat 3) the Coll(E) promoter-enhancer may be useful to take advantage ofthe levels of cytokines found in the brain after grafting.

The observation that cytokines can downregulate the expression from theLTR suggests that fibroblasts implanted into the brain may be affectedby cytokines that are produced by blood-borne mononuclear phagocytes andlymphocytes that have penetrated into the brain after grafting.Microglial and endothelial cells, another source of cytokines in thebrain, may contribute to this scenario. Thus, expression of LTR-driventransgenes in grafted fibroblasts may eventually be compromised withtime post-transplantation. One way to counteract the detrimental actionsof cytokines on the LTR is to use molecules such as anti-inflammatoryagents that curtail the production of cytokines. This treatment may notonly increase the stability of transgene expression in implantedfibroblasts, but may contribute to the survival of the cells by reducingthe immune response.

An alternative approach takes advantage of cytokines by using a promoterthat is up-regulated, or at least not affected, by cytokines. Thisexample demonstrates that TGFβ1 and TNFα do not negatively affecttransgene expression (as assessed by CAT activity) inColl(E)-fibroblasts. In comparison, TNFα increases expression. Infγ hada similar effect on the Coll(E) promoter-enhancer as it did on the LTR(i.e. reduction of transgene expression). Therefore, it may still benecessary to control the release of cytokines such as Infγ fromT-lymphocytes, with immunosuppression drugs such as cyclosporin.

The data also indicates that expression from the Coil(E)promoter-enhancer is significantly increased in cells maintained in 2%as compared to 10% serum-containing medium. The level of expression iscomparable to that observed in fibroblasts containing the LTR.Fibroblasts implanted into the body would be exposed to less serumelements than in culture. Therefore, expression from the Coll(E)promoter-enhancer under certain conditions may be equal to the strongestviral promoters currently used to drive transgene expression.

EXAMPLE X Regeneration of Adult Axotomized Neurons

In this Example, a new in vivo model is presented to assess thesubstrate that supports aberrant axon growth in response to trophicfactors in the adult rat CNS. In addition, the importance of NGF on theregenerative capacities of rat septal neurons after axotomy is assessed.

This Example demonstrates that adult reactive astrocytes can serve aspermissive substrates for axon growth when supplied by NGF fromintrastriatal grafts of genetically modified primary fibroblasts. Inaddition, the effects of grafts of modified primary fibroblastssecreting NGF on the regenerative capacities of rat septal neurons afteraxotomy are assessed.

Grafts consisting of primary skin fibroblasts genetically modified toexpress and secrete NGF prepared as described above in Example II wereimplanted into the striatum of adult rats. Primary fibroblasts wereobtained from a skin biopsy of a female Fischer 344 rat. The cells weremaintained under standard culture conditions and fed DMEM containing 10%fetal calf serum three times a week. Primary cells were infected with amurine retroviral vector containing the cDNA for mouse β-NGF, pLN.8RNL,as described above in Example II; infected cells were selected with theneomyocin analogue G418. Prior to isolating the cells for grafting,samples of culture media were taken from control and infected cells.Employing a sensitive two-site immunoassay (Boerhinger-Mannheim), it wasdetermined that the infected cells produced and secreted 154-173 pgNGF/hr/10⁵ cells; levels of NGF could not be detected in control media.Female Fischer rats (weighing approximately 175-200 g) were anesthesizedwith a mixture of ketamine-xylazine (ketamine, 25 mg/ml; rompun, 1.3mg/ml; and acepromazine, 0.25 mg/ml). The heads were shaved and placedin a stereotaxic frame; antiseptic was applied to the head beforesurgery commenced. Each animal received 3×10⁵ cells in 3 μl of graftingsolution (phosphate buffered saline supplemented with 1 μg/ml of MgCl₂and CaCl₂, and 0.1% glucose) into the striatum: NGF-producing cells intothe right, and non-infected cells into the left. After survival periodsof one, three and eight weeks, the animals were anesthetized andperfused transcardially with 4% paraformaldehyde and 0.1% glutaraldehydein 0.1M phosphate buffer. Horizontal or sagittal sections through graftswere cut on freezing microtome and processed for NGF receptorimmunoreactivity, or were cut on a vibratome and processed for electronmicroscopic examination.

Genetically modified cells were injected into the right striatum andnon-infected control cells into the left striatum. One week afterimplantation, a plexus of NGF receptor-immunoreactive axons was evidentat the caudal pole of striatal grafts composed of NGF-producingfibroblasts, whereas only a small number of enlarged and swollenimmunoreactive axonic profiles were observed adjacent to grafts ofnon-infected control cells (FIG. 49a and 49b). At three weeks, thedensity of immunoreactive axons surrounding the NGF-producing graftsincreased dramatically; a few NGF receptor-immunoreactive profilescompletely filled the grafts composed of NGF-producing cells, and theplexus of axonic profiles surrounding the grafts was no longer present(FIG. 49d). These immunoreactive profiles appeared larger than a singleaxon and usually extended in a vertical fashion. Although grafts ofnon-infected primary fibroblasts lacked such patterns of immunostainedaxonic profiles at three and eight weeks after implantation, NGFreceptor immunoreactivity was confined to a few blood vessels (FIG.49c).

Ultrastructural examination revealed that all intrastriatal grafts werecomposed of primary fibroblasts with extensive rough endoplasmicreticulum and an extracellular matrix filled with collagen (FIG. 50).Capillaries composed of nonfenestrated endothelial cells contributed tothe extensive vascular plexus within these grafts (FIG. 50). At threeand eight weeks after implantation, reactive astrocytes completelyenveloped the grafts; these astrocytic profiles possessed a distinctbasal lamina on those surfaces apposing the graft environment. Thisbarrier between the CNS neuropil and non-CNS graft was characteristic ofa "glial scar" usually found following penetrating wounds of theneuropil. Also, many processes of reactive astrocytes extended into thesubstance of the grafts and were often closely associated withcapillaries. Within the extracellular matrix of the NGF-producing graftsonly, bundles of unmyelinated axons were interspersed throughout thegrafts; terminals containing clear, spherical vesicles were occasionallyobserved as well. Reactive astrocytic processes laden with filamentswere found to envelope these axonic profiles in a convoluted fashion(FIG. 51a and 51b). Basal lamina surrounded the entire axo-glial complexand was not evident on opposing plasma membranes between axons andastrocytes. The incidence of these axo-glial arrangements was greater ateight weeks than at three weeks following implantation. Axonic profileswere not found within grafts of non-infected control cells.

These data reveal that NGF receptor-positive axons grow toward grafts ofNGF-producing primary skin fibroblasts. Grafts of non-infected cells, onthe other hand, do not elicit a similar neuronal response. Implants ofboth types of cells did, however, induce reactive astrocytes to surroundand encapsulate the grafts before three weeks. Despite the presence ofthis glial barrier, unmyelinated axons migrating along elaboratearrangements of reactive astrocytic processes were found within theNGF-producing grafts only; these axons were confined exclusively toastrocytic surfaces that lacked a distinct basal lamina. Although theextracellular matrix of primary fibroblast implants consists of otherconducive substrates for axon growth, e.g. collagen, laminin andfibronectin, growing axons within the grafts were not associated withthese components, all of which have been previously demonstrated asconducive substrates for axon growth in vitro. Thus, for NGF-sensitiveaxons penetrating grafts of genetically modified primary skinfibroblasts, reactive astrocytes are the preferred substrate for axonelongation.

These results show that mature reactive astrocytes in the adult CNS canact as substrates which promote rather than inhibit axon growth.Employing intrastriatal grafts of genetically modified cells thatproduce NGF, these data show that successful axon elongation requiresthe necessary tropic and trophic support. Thus, it appears that theavailability of growth-promoting factors, and not the substrates forgrowth, is a critical influence during regeneration.

To examine the effects of grafts on regeneration of axons, primary cellsgenetically modified to express NGF in vivo were established and shownto provide trophic support of axotomized cholinergic septal neurons. Toprovide a cellular source of NGF in vivo, primary skin fibroblastsgenetically modified to express NGF prepared as described in Example II,were suspended in a collagen matrix and placed in the cavity formed by aunilateral ablation of the fimbria-fornix (FF) pathway, a major routefor septal axons projecting to the rostral hippocampus.

Under anesthesia, primary skin fibroblasts were obtained from biopsiesof the ventral abdominal wall of a female Fischer 344 rat and thesecells were maintained under standard culture conditions. Using aretroviral vector containing the cDNA for mouse β-NGF, pLN.8RNL, primarycells were infected in culture, and NGF production by these cells waslater assessed using a two-site immunoassay (also as described inExample II). To suspend the cells in a collagen matrix, cultures ofNGF-producing and control non-infected primary cells were rinsed inphosphate-buffered saline, trypsinized and suspended in DMEM. The cellswere then counted and a total of 10⁶ cells were aliquoted in medium.Type I collagen from rat tail (Sigma) was dissolved with 0.1% aceticacid for a final concentration of 3% collagen. Sodium hydroxide (0.1N)was added to the cell suspension to make the medium more alkaline, andthe collagen was then added (final concentration of 1%), mixed andaliquoted into centrifuge tubes. The collagen/fibroblast matrices werethen incubated at 37° C. for 48 h. Forty female Fischer 344 rats(weighing approximately 175 g) were anesthetized for surgery using amixture of ketamine (75 mg/kg), Rompun (4.0 mg/kg) and acepromazine (5.6mg/kg). Their heads were shaved, and the animals were placed in a Kopfstereotaxic frame. Surgery was a two-step procedure as described inExample II. First, unilateral aspiration lesions through the cingulatecortex and the FF pathway were performed under stereoscopic vision.Then, pieces of the collagen/fibroblast matrices were cut into smallpieces (approximately 2-3 mm³) and placed into the wound cavity; otheranimals received only FF lesions. The animals recovered from surgery andsurvived for three, four or eight weeks. After these survival periods,the animals were deeply anesthetized, as described above, and wereperfused transcardially with 4% paraformaldehyde in phosphate buffer(0.1% glutaraldehyde was added for ultrastructural cases). The brainswere removed, post-fixed, cut on either a freezing microtome orvibratome, and the sections (40 to 50 μm in thickness) were processedfor acetylcholinesterase histochemistry, immunohistochemistry andultramicrotomy.

Only those animals that received a complete unilateral FF lesion andpossessed a graft within the cavity were assessed for: 1) septalneuronal savings following axotomy; 2) acetylcholinesterase (ACHE)histochemical and NGF receptor immunohistochemical staining in thegrafts and hippocampus; 3) Nissl staining and tyrosine hydroxylase (TH),glial fibrillary acidic protein (GFAP), and laminin immunohistochemicalstaining in the grafts; 4) retrograde transport of fluorescent markersfrom the hippocampus ipsilateral to the lesion and grafts of eitherNGF-producing or control non-infected fibroblasts; 5) ultrastructuralorganization of the grafts; and 6) ultrastructural distribution of AChEstaining in the deafferented dentate gyrus ipsilateral to grafts ofNGF-producing grafts.

Histochemistry and immunohistochemical detection were performed asfollows. Alternate sections through collagen/fibroblast grafts werestained for Nissl substance using aqueous 0.5% thionin, stainedhistochemically for AChE using a modified method of Hedreen et al., J.Histochem. Cytochem. 33: 134-140 (1985), and stainedimmunohistochemically for laminin, glial fibrillary acidic protein(GFAP) and tyrosine hydroxylase (TH). Sections through the septum werestained immunohistochemically for NGF receptor, and sections through thehippocampus were stained histochemically for AChE andimmunohistochemically for NGF receptor and TH.

Immunohistochemical detection of NGF receptor, TH and GFAP used the sameprotocol. Sections were initially treated in a solution of 0.6% aqueoushydrogen peroxide for thirty minutes, briefly rinsed in TBS andincubated in a solution containing TBS, 3% normal horse serum and 0.25%Triton-X for one hour (this solution was used to dilute all antibodies).The sections were then incubated for 48 hours at 4° C. in a solutioncontaining one of the following antibodies: monoclonal 192 IgG to NGFreceptor, (Chandler et al., J. Biol. Chem. 259: 6882-6889 (1984, 1:100dilution), monoclonal IgG to TH (Boehringer-Mannheim; 1:250) andmonoclonal IgG to GFAP (Amersham, 1:100). Control sections wereincubated in solutions lacking primary antisera. All sections were thenrinsed in buffer, incubated in biotinylated horse anti-mouse IgG (1:167)for one hour at room temperature. After another rinse, the sections wereincubated in avidin-biotin complex (ABC; Vector Laboratories) for onehour. They were then rinsed again and reacted in a solution containing0.025% diaminobenzidine tetrahydrochloride, 0.5% nickel chloride, and0.18% hydrogen peroxide in TBS for five minutes. The reaction wasstopped by rinsing the sections in buffer.

The sections through the grafts were also stained immunohistochemicallyfor laminin. Again, the sections were initially treated with 0.6%aqueous hydrogen peroxide, rinsed, and incubated in a solution of TBS,3% normal goat serum and 0.25% Triton-X. The sections were thenincubated in the same solution as above with the addition of rabbitanti-human laminin IgG (1:800 dilution) for 48 hours at 4° C.; controlsections were incubated in a solution lacking the primary antiserum.They were then rinsed and incubated in biotinylated goat anti-rabbit(1:220) for one hour at room temperature. After another rinse, thesections were incubated in ABC and reacted as above.

Three (3) and eight (8) weeks after unilateral ablation of the FF andplacement of grafts composed of collagen and primary fibroblasts, amarked decrease in the population of NGF receptor-immunoreactive neuronswas evident in the ipsilateral medial septum (FIG. 52a and 52b). Thelines drawn through the medial septum (MS) and graft (G) and hippocampaldentate gyrus (DG) represent the level of coronal sections ofphotomicrographs in this illustration. Injection sites for thefluorescent microspheres within the hippocampus are also shown. A modestsavings of NGF receptor-positive neurons in the ipsilateral (right)septum is evident with NGF-producing grafts (arrowheads indicate septalmidline).

For determination of NGF receptor-immunoreactive septal neurons savedwith grafts of NGF-producing fibroblasts, five sections, 120 μm apart,through the septal area stained immunohistochemically for NGF receptorwere selected for neuronal cell counting. A counting grid (0.5×0.5 mm)was used to count all immunoreactive neurons in the ipsilateral andcontralateral medial septum. Differences between the percentage of NGFreceptor-positive surviving cells per animal for each group of animalswere assessed by a one-way ANOVA. The results are summarized in Table 4,showing percentages of septal neurons saved (contralateral vs.ipsilateral) with grafts of NGF-producing fibroblasts or controlnon-infected fibroblasts in the lesion cavity, or without grafts, three(or four) and eight weeks following unilateral FF ablation. Coronalsections through the septum were stained immunohistochemically for NgFreceptor to evaluate the survival of septal neurons. Sections throughthe fibroblast grafts were stained immunohistochemically for TH toassess sympathetic axon ingrowth, for GFAP to assess astrocyticresponses to the grafts, and for laminin to reveal neovascularization.Sections through the grafts and ipsilateral hippocampus were stainedhistochemically for AChE and NGF receptor to examine the regrowth ofseptal axons.

                  TABLE 4                                                         ______________________________________                                                     Post-operative survival periods                                               3 (or 4) weeks                                                                           8 weeks                                               ______________________________________                                        NGF-producing grafts                                                                         68 ± 4* (n = 3)                                                                         62 ± 7* (n = 7)                                Control grafts 47 ± 4  (n = 4)                                                                         47 ± 5  (n = 8)                                No grafts      44 ± 8  (n = 3)                                                                           --                                              ______________________________________                                         *Significance at the P < 0.01 level                                      

Table 4 shows that grafts of NGF-producing primary fibroblasts sustainedsignificantly higher percentages of NGF receptor-positive septal neuronsat three (or four) and eight weeks than did grafts of collagen withcontrol non-infected primary fibroblasts at the same time periods. Thepercentages of NGF receptor-immunoreactive neurons are comparable withcontrol non-infected grafts and without grafts. Thus, while the degreeof savings with NGF-producing cells was modest, these results provideindirect evidence for the in vivo expression of the transgene for NGF bygenetically modified primary fibroblasts. This expression should enablethe cells to provide trophic support to axotomized cholinergic septalneurons.

Regeneration of septal axons within the grafts and deafferentedhippocampal dentate gyrus was assessed through the detection of AChE andNGF receptor staining. Grafts of NGF-producing fibroblasts usuallystained moderately to intensely for AChE (FIG. 52c). Grafts of control,non-infected primary fibroblasts, on the other hand, often lacked AChEreactivity (FIG. 52d). Note that the NGF-producing graft possesses adensely stained perimeter, whereas the control graft lacks AChEreactivity (dotted line represents approximate boundary of the graft).

Three weeks after the transection of the FF unilaterally and placementof control, non-infected fibroblasts/collagen grafts within the cavity,the deafferented dentate gyrus was devoid of axons stained for AChE andNGF receptor. This was in stark contrast to the prominent staining forAChE (FIG. 52e) and NGF receptor seen in the normal dentate gyrus. Thenormal pattern of AChE staining reveals a robust input to all layers ofthe dentate gyrus. By eight weeks after lesion/implantation, thedeafferented dentate gyrus still lacked AChE-positive axons (FIG. 52g)but now possessed large-diameter axons stained immunohistochemically forNGF receptor, particularly in the polymorphic layer of the dentategyrus; these axons are reminiscent of sympathetic axons that sprout intothe hippocampus following FF lesions (see Batchelor et al., J. Comp.Neurol. 284: 187 (1989)). In two cases, ACHE-positive axons wereobserved traversing beneath the control grafts from the contralateral tothe deafferented hippocampus. Three weeks after the unilateraltransection of the FF and placement of NGF-producing fibroblasts incollagen, a moderate plexus of AChE-positive axons was evident in themedial and rostral aspects of the deafferented dentate gyrus. By eightweeks, the density of AChE-positive fibers increased markedly, yet mostaxons were still confined to the rostromedial dentate gyrus. In certaincases, however, a robust plexus of topographically organizedAChE-positive axons was observed within the dentate gyrus and CA2-3fields (FIG. 52f). Although the pattern of reinnervation was comparableto the distribution of AChE-positive septal axons in the normalundamaged hippocampus (FIG. 52e), the density of axons was usually less.In those cases with NGF-producing grafts, large-diameter axons stainedfor NGF receptor were also observed in the polymorphic layer of thedeafferented dentate gyrus, usually in the caudal portions of thehippocampus.

Although the pattern of AChE staining within the NGF-producing andcontrol grafts was strikingly different, both types of grafts possessedsimilar cellular populations and immunostaining for TH, GFAP andlaminin. Nissl staining showed that all grafts were composed of densebundles of collagen with a variety of different cell types within andsurrounding the grafts, including fibroblasts, lymphocytes, mast cells,plasma cells, astrocytes and endothelial cells of capillaries. Also,both types of grafts possessed a small number of TH-immunoreactiveaxons, particularly within the dense collagen bundles. Astrocyticprocesses stained immunohistochemically for GFAP were seen extendingfrom the damaged neural tissue around the wound cavity and penetratinginto the outer aspects of the grafts; occasionally, GFAP-positive cellswere seen within the grafts as well. Laminin immunostaining of grafts ofNGF-producing and non-infected primary fibroblasts revealed an extensivehost-derived vascular network. Also, elongated laminin-immunoreactivecell bodies, perhaps Schwann cells, were found within and around thedense collagen bundles of both types of grafts.

To determine the origin of those septal neurons that regenerate newaxons following FF ablation and reinnervate the deafferentedhippocampus, stereotaxic placements of fluorescent microspheres weremade into the ipsilateral dentate gyrus and CA2-3 fields. Nine (9)deeply anesthetized animals with grafts of either NGF-producing (n=5) orcontrol, non-infected (n=4) fibroblasts received two stereotaxicplacements of rhodamine-fluorescent microspheres (200 nl/site) into thedentate gyrus and CA2-3 fields in the hippocampus ipsilateral to thelesion and graft eight weeks after surgery. Following a 48 h recovery,the animals were again anesthetized and perfused transcardially with 4%paraformaldehyde. The brains were sectioned coronally on a freezingmicrotome, and retrogradely labeled neurons within the septal areas wereviewed with a fluorescence microscope. The schematic illustration inFIG. 52 shows the injection sites.

Most retrogradely fluorescently labeled elongated neurons were foundwithin the ipsilateral medial septum and diagonal band areas, while asmaller number were observed in the septal midline and in thecontralateral septal nuclei (FIG. 53). Cases with grafts of control,non-infected fibroblasts did not possess retrogradely labeled neurons inany area of septal region.

At the ultrastructural level, the grafts consisted of dense collagenbundles with a loose outer reticular arrangement eight weeks afterimplantation. Fibroblasts were observed both within the dense andreticular collagenous formations, and attenuated processes of thesecells enveloped dense accumulations of collagen in both areas.Capillaries composed of non-fenestrated endothelial cells surrounded bybasal lamina were found throughout the grafts. Several types of cellswere also observed within the grafts, including lymphocytes, plasmacells, mast cells, and phagocytes. Astrocytes and their processes werefound predominantly within the loose reticulum around the dense collagencenter. The most prominent difference between grafts of NGF-producingand non-infected fibroblasts was the number of unmyelinated axons, i.ewithin an area of 960 μm², 1625 axons were found in the NGF-producinggrafts compared with 329 axons in the control grafts. Ultrathin sectionsof either NGF-producing (n=3 different cases) or control,. non-infected(n=3 different cases) fibroblast/collagen grafts were examined and thetotal number of axons were counted in 10 grid squares (2 parallelcolumns of 5 grid squares, a total area of 960 μm², with graft tissue ofthe dense and loose collagen areas). Among the three cases ofNGF-producing grafts, 1625 axons were found in comparison to 329 axonsamong the three cases of control, non-infected grafts.

As shown in FIGS. 54a-FIG. 54c large numbers of axons were surrounded byor passed along narrow processes of astrocytes (*) that containintermediate filaments. These astrocytic profiles possess basal lamina(arrowheads) on those cell surfaces facing the graft extracellularmatrix. The axons are usually found apposing those astrocytic surfaceslacking a basal lamina. Such axons were also found near the cell bodiesof the astrocytes and other cells, including fibroblasts (F) (FIG. 54d)and other astrocyte-like cells and their processes. Still otherunmyelinated axons were closely associated with the basal lamina of theendothelial cells of capillaries (C) or found within the extracellularmatrix of the NGF-producing grafts; collagen fibrils are distributedamong the axons (FIG. 54e). Within grafts of either NGF-producing orcontrol fibroblasts, small numbers of unmyelinated axons enveloped bySchwann cells (S) and their processes were observed within the reticularareas (L=lumen of capillary) (FIG. 54f). Axons observed within the densecollagen area of both types of grafts were usually ensheathed withinattenuated glial profiles. Occasionally, control grafts also possessed avery small population of axons within the extracellular matrix.

Ultrastructural examination of the deafferented hippocampus followingthe placement of NGF-producing grafts revealed a sparse distribution ofAChE reactivity within the dentate neuropil. The electron-dense reactionproduction was localized to the plasma membranes of small-diameter,unmyelinated axons and terminal containing clear spherical vesicles;these stained axonic profiles were found predominantly in the molecularlayers of the dentate gyrus (FIG. 55). Dispersed throughout the dentateneuropil were clusters of AChE-positive unmyelinated axons, and theseaxonal aggregates were usually found near astrocytic processes.Occasionally, axon terminals possessing reactivity for AChE formedsynaptic contacts with dendritic shafts and spines. The symmetry ofcontacts between AChE-positive terminals and dendritic profiles ofgranular neurons, however, was usually obscured by the localization ofreaction product at the site of apposition. Since sections of thedeafferented dentate gyrus of those animals with control non-infectedcells lacked AChE reactivity, further analysis was not conducted at theultrastructural level.

In particular, FIG. 55 shows a coronal section (40 μm) of the dentategyrus stained for ACHE, taken immediately adjacent to that tissueexamined at the ultrastructural level. ACHE-positive fibers are evidentin the inner (IM) and outer (OM) molecular layers and the granular layer(G). AChE-positive somata and fibers are also found in the polymorphiclayer (P). FIG. 55b and 55c show that clusters of AChE-positiveunmyelinated axons are found throughout the granular (B) and molecular(C) layers. FIG. 55d, 55e and 55f show that ACHE-positive terminals formsynaptic contacts (arrowheads) with dendritic shafts (Sh) and spines(Sp) in the molecular layers. Astrocytic processes (As) are usuallyobserved near clusters of AChE-positive axons and terminals in thedentate gyrus.

These results demonstrate the importance of trophic/tropic substancessuch as NGF in the regenerative capacity of septal cholinergic neurons,as assessed by the percentage of NGF receptor-positive neurons savedwithin the medial septum and the reinnervation of the hippocampaldentate gyrus following a unilateral FF lesion and placement of graftscomposed of a collagen matrix with NGF-producing primary fibroblasts.Intracerebral grafts of NGF-producing primary fibroblasts are thuscapable of preventing the retrograde degeneration of cholinergic septalneurons after axotomy like grafts of NGF-producing immortalizedfibroblasts as shown in Example II herein, and infusions of exogenousNGF into the lateral ventricle (Hefti, J. Neurosci. 6: 2155 (1986);Kromer, Science 235: 214 (1987) and Williams et al., Proc. Natl. Acad.Sci. USA 83: 9231 (1986)). The percentages of cells saved withNGF-producing primary fibroblasts in a collagen matrix, however, is muchlower than that achieved with intraventricular infusions of NGF orimplants of NGF-producing immortalized fibroblasts. This may be dueto 1) a small number of cells actually implanted within the collagenmatrix resulting in a lower level of NGF production; and 2) a possibledown-regulation of the transgene following implantation.

These results show that, like other different types of tissues whichsupport new axon growth from septal neurons, grafts of NGF-producingfibroblasts within a collagen matrix also support the growth of septalfibers, such that a sparse reinnervation of the hippocampus is evidentat three weeks. By eight weeks, however, the density of axons within thehippocampus is clearly greater than that seen at three weeks. Implantsof control cells in collagen offer little or no support, since theingrowth of septal AChE-positive fibers to the hippocampus is negligibleand no labeled cells are found in the septal area ipsilateral to thedentate gyrus following placements of retrograde tracer. Likewise,grafts of acellular peripheral nerve (i.e., those lacking Schwann cells)cannot support the growth of septal axons (Hagg et al., Exp. Neurol.112: 79 (1991)). Together, these data reveal that septal axons are ableto use many different graft environments, consisting of both cellularand extracellular substrates for growth.

These data also show that collagen grafts containing primary skinfibroblasts induce a similar ingrowth of capillaries with basal lamina,astrocytes and Schwann cells, regardless of whether the primary cellsare genetically modified to produce NGF or not. Moreover, theextracellular matrices of both types of grafts are similar, such thatthe patterns of laminin immunostaining and collagen distribution arecomparable. The real differences between NGF-producing grafts andcontrol grafts lie in the robust staining for AChE and abundance ofunmyelinated axons within the NGF-producing grafts. First, prominentAChE reactivity is only evident in NGF-producing grafts; control graftsusually lack AChE staining. Both types of grafts, however, possess asparse population of TH-immunoreactive fibers, as well as axonsenveloped within Schwann cells and their processes; most axonsassociated with these glial elements may represent TH-positivesympathetic ingrowth. Second, NGF-producing grafts possess numerousaxons that are ensheathed by astrocytic processes, pass along the basallamina of capillaries and astrocytes, or extend within the loosearrangements of collagen. Control grafts, on the other hand, onlypossess a very small population of axons within the extracellularenvironment. From these collective data, it appears that NGF-sensitiveaxons arising from perturbed septal neurons require the availability ofNGF and a permissive graft environment for new growth. Using grafts ofgenetically modified cells that produce NGF in vivo regenerating septalaxons have been shown herein to grow on a variety of differentsubstrates only in its presence. Without elevated levels of NGF, axonsdo not regenerate in response to grafts consisting of collagen andcontrol non-infected fibroblasts, even though the same cellular andextracellular substrates are available.

Previous studies examining the regenerative capacity of septalcholinergic neurons in the rat following axotomy and grafting havereferred to the "reinnervation" of the deafferented hippocampus withcertain bridging environments (Hagg et al., Exp. Neurol. 112: 79(1991)). To date, however, no investigation has provided directmorphological evidence that AChE-positive septal axons within thehippocampus actually innervate postsynaptic targets. Ultrastructuraldata presented herein reveal that axons stained for AChE activity aresparsely distributed within the deafferented dentate gyrus eight weeksafter FF lesion and grafting of NGF-producing fibroblasts. These axonsare often seen in clusters of two or more. The ultrastructuralorganization of AChE-positive septal axons is similar to recentobservations that septal axons stained immunohistochemically for NGFreceptor also form small aggregates within the normal rat dentate gyrus(Kawaja and Gage, J. Comp. Neurol. 307: 517 (1991)). In addition, thesenewly-formed AChE-positive axons give rise to terminals that formsynaptic contacts with dendritic shafts and spines in the deafferenteddentate gyrus. The pattern of synaptic contacts between septalcholinergic axons and granular dendritic profiles is comparable to thatnormally found in the rat dentate gyrus; axosomatic contacts betweencholinergic axons and granular neurons are rare (Shute and Lewis,Zellforsch. Mikrosk. 69: 334.(1966); and Clarke, Brain Res. 360: 349(1985)). It is worth noting that when grafts of fetal septum areimplanted within the adult rat dentate gyrus after FF ablation,cholinergic axons from the grafts innervate the somata of granularneurons at higher frequency than normal (Clarke et al., Brain Res. 369:151 (1986)). Further, the number of graft-derived cholinergic axonscontacting dendritic profiles decreases dramatically (Id). From thesedata, it appears that while the cholinergic axons from fetal septalgrafts within the deafferented dentate gyrus form unique synapticarrangements with granular neurons, septal axons that regenerate acrossNGF-rich grafts of collagen and fibroblasts recapitulate a normalsynaptic organization within the dentate gyrus (i.e., AChE-positiveseptal axons terminate predominantly on dendritic shafts and spines).

The results presented in this example demonstrate that grafts ofNGF-producing fibroblasts in a collagen matrix sustain damagedcholinergic neurons of the rat medial septum, provide a conduciveenvironment for regrowing NGF-sensitive axons arising from these neuronsand influence the reinnervation of the deafferented granular neurons ofthe dentate gyrus by septal axons. In particular, these grafts induceaxonal elongation and synaptic connectivity. Regenerating NGF-sensitiveseptal axons use both cellular and matrix substrates for growth withingrafts consisting of NGF-producing primary fibroblasts and collagen.Without elevated levels of NGF (i.e. using control fibroblast/collagengrafts), a higher proportion of septal neurons undergo degeneration andaxons fail to grow, despite the presence of conducive substrates.

In addition to NGF, other neurotrophic factors, such as brain-derivedgrowth factor (BDNF), neurotrophin (NT)-3, NT-4, and ciliary neuronaltrophic factor (CNTF), may be used to sustain axotomized neurons andpromote axon regrowth of other neuronal populations in the CNS.

It is apparent that many modifications and variations of this inventionas set forth above may be made without departing from the spirit andscope of the present invention. The specific embodiments described aregiven by way of example only and the invention is limited only by theterms of the appended claims.

We claim:
 1. A method for treating defective, diseased or damaged cellsin the mammalian central nervous system comprising grafting donor cellsinto the central nervous system of a subject, said subject or donorcells treated so as to minimize graft rejection, said donor cellsgenetically modified by insertion of at least one transgene encoding aproduct or products which directly or indirectly affect the cells intosaid cells to produce functional molecules in a sufficient amount toameliorate said defective, diseased or damaged cells in the centralnervous system.
 2. The method of claim 1, wherein the step of graftingsaid donor cells comprises introducing said donor cells into the brainof a subject.
 3. The method of claim 1, wherein the step of graftingsaid donor cells comprises introducing said donor cells into the spinalcord of a subject.
 4. The method of claim 2, wherein said introducingcomprises intracerebral, intraventricular, subdural space, putamen,nucleus basalis, hippocampus, cortex, striatal, caudate and intravenousintroduction.
 5. The method of claim 1, wherein said transgene iscarried by a viral vector.
 6. The method of claim 5, wherein said vectoris a herpes virus vector.
 7. The method of claim 5, wherein said vectoris a neurotropic virus vector.
 8. The method of claim 5, wherein saidvector is a retrovital vector.
 9. The method of claim 8, wherein saidretroviral vector is the retroviral vector pLN.8RNL having a finalconstruction as shown in FIG.
 12. 10. The method of claim 8, whereinsaid retroviral vector is the retroviral vector pLThRNL having a finalconstruction as shown in FIG.
 16. 11. The method of claim 1, wherein thetransgene is inserted into donor cells by a nonviral physicaltransfection of DNA encoding a transgene.
 12. The method of claim 11,wherein said nonviral physical transfection comprises microinjection ofDNA encoding a transgene.
 13. The method of claim 1, wherein thetransgene is inserted into donor cells by electropotation.
 14. Themethod of claim 1, wherein the transgene is inserted into donor cellschemically mediated transfection.
 15. The method of claim 14, whereinsaid chemically mediated transfection comprises calcium phosphatetransfection.
 16. The method of claim 1, wherein the transgene isinserted into donor cells by liposomal mediated transfection.
 17. Themethod of claim 1, wherein said step of insertion into donor cellscomprises lipofection.
 18. The method of claim 1, wherein said moleculesare selected from the group consisting of growth factors, enzymes,gangliosides, antibiotics, neurotransmitters, neurohormones, toxins,neurite promoting molecules, antimetabolites and precursors of saidmolecules.
 19. The method of claim 1 further comprisingco-administration of a therapeutic agent for treating said disease ordamage to the central nervous system.
 20. The method of claim 19,wherein said therapeutic agent is selected from the group consisting ofgrowth factors, gangliosides, antibiotics, neurotransmitters,neurohormones, toxins, antimetabolites, neurite promoting molecules andprecursors of these agents.
 21. The method of claim 19, wherein saidtherapeutic agent is cellular matter.
 22. The method of claim 21,wherein said cellular matter is selected from the group consisting ofadrenal chromaffin cells, fetal brain tissue cells and placental cells.23. The method of claim 1 further comprising implanting material to thesite of said damage or disease, said material to facilitate reconnectionor ameliorative interactions of injured neurons.
 24. The method of claim23, wherein said material is selected from the group consisting ofhomogenate of brain, homogenate of placenta, collagen, whole cells,synthetic material, neurite promoting extracellular matrix, andgenetically modified donor cells.
 25. The method of claim 1, whereinsaid donor cells are selected from the group consisting of fibroblasts,neurons, glial cells, keratinocytes, hepatocytes, ependymal cells, bonemarrow cells, hippocampal cells, olfactory mucosa cells, stem cells,adrenal cells, connective cells, leukocytes and chromaffin cells. 26.The method of claim 1, wherein said donor cells comprise a mixture ofcell types from different anatomical regions.
 27. The method of claim 1,wherein said cells are primary cells.
 28. The method of claim 1, whereinsaid cells are immortalized cells.
 29. The method of claim 1, whereinthe step of grafting said donor cells comprises grafting fromapproximately 10⁴ to approximately 10⁸ cells per graft.
 30. The methodof claim 1, wherein said donor cells consist of cells that have beenpassaged from approximately 2 to approximately 20 times.
 31. The methodof claim 1, wherein the step of grafting comprises multiple grafting ofgenetically modified donor cells in several different sites.
 32. Themethod of claim 31, wherein said donor cells comprise a mixture oftransgenes inserted into said cells.
 33. The method of claim 31, whereinsaid donor cells comprise a mixture of cell types.
 34. The method ofclaim 1, wherein the step of grafting comprises multiple grafting ofgenetically modified donor cells into a single site.
 35. The method ofclaim 34 wherein said donor cells comprise a mixture of cell types. 36.The method of claim 5, wherein said vector carries a promoter to enhanceexpression of said transgene when said donor cells are quiescent. 37.The method of claim 36, wherein said promoter is a collagen promoter.38. The method of claim 37 wherein said collagen promoter is selectedfrom the group consisting of α1(I) and α2(I).
 39. The method of claim36, wherein said vector further carries an enhancer sequence to increasethe activity of said promoter.
 40. The method of claim 39, wherein saidenhancer sequence is SV40 enhancer sequence.
 41. The method of claim 39,wherein said enhancer sequence is a collagen promoter enhancer sequence.42. The method of claim 41, wherein said enhancer sequence is a α2(I)collagen enhancer sequence.
 43. The method of claim 36 furthercomprising the administration of cytokines toregulate the expression ofsaid molecules.
 44. The method of claim 43, wherein said cytokines areselected from the group consisting of interleukin-1β, interferon-α,tumor necrosis factor-α, tumor growth factor-β, basic fibroblast growthfactor (bFGF) and epidermal growth factor (EGF).
 45. The method of claim1 further comprising the use of growth factors to maintain survival ofthe donor cells in the mammalian CNS.
 46. The method of claim 45,wherein said growth factors are bFGF or EGF.
 47. The method of claim 1further comprising regulation of the secretion of said functionalmolecules by administration of a precursor for said molecules.
 48. Themethod of claim 47, wherein said functional molecules are acetylcholine,and said precursor is choline.
 49. The method of claim 1, wherein saiddonor cells are primary fibroblasts.
 50. The method of claim 49, whereinsaid donor cells are primate fibroblasts.
 51. The method of claim 49,wherein said donor cells are human fibroblasts.
 52. A method fortreating defective, diseased or damaged cells in the central nervoussystem of a mammalian subject comprising:a) grafting fibroblasts from amammal, genetically modified with a vector containing ChAT cDNA toexpress acetylcholine into the central nervous system of the mammalhaving defective, diseased or damaged cells in the central nervoussystem said subject or fibroblasts treated so as to minimize graftrejection; and b) administering choline chloride orally to maintain andenhance secretion of said acetylcholine from said fibroblasts.
 53. Themethod of claim 52, wherein said method is used to treat a patientsuffering from Alzheimer's disease.
 54. A method for enhancing theexpression of a therapeutic transgene product from quiescent donor cellsgrafted into a central nervous system of a mammalian subject, said donorcells genetically modified by insertion of a therapeutic transgene via avector carrying said transgene, said subject or donor cells treated soas to minimize graft rejection, the method comprising inserting apromoter in said vector.
 55. The method of claim 54, wherein saidpromoter is a non-viral promoter.
 56. The method of claim 55, whereinsaid promoter is a collagen promoter.
 57. The method of claim 56,wherein said promoter is selected from the group consisting of α1(I)collagen and α2 (I) collagen promoters.
 58. The method of claim 54,wherein the expression of said therapeutic transgene is further enhancedusing an anti-inflammatory agent to reduce the production of cytokines.59. The method of claim 58, wherein said anti-inflammatory agent isasteroid.
 60. The method of claim 59, wherein said anti-inflammatoryagent is dexamethasone.
 61. The method of claim 54 further comprisingthe use of an immunosuppression agent to reduce the production ofcytokines.
 62. The method of claim 61, wherein said immunosuppressionagent is cyclosporin.
 63. The method of claim 61, wherein said cytokinesare Infγ.
 64. A method for treating defective, diseased or damaged cellsin the central nervous system of a mammalian subject comprising graftinggenetically modified primary fibroblasts into the central nervoussystem, said fibroblasts modified by insertion of a therapeutictransgene carried in a retrovital vector to produce the product the ofsaid defective diseased or ed cells, said subject or fibroblasts treatedso as to minimize graft rejection.
 65. The method of claim 64, whereinsaid vector is the retroviral vector pLN.8RNL having a finalconstruction as shown in FIG.
 12. 66. The method of claim 64, whereinsaid vector is the retroviral vector pLThRNL having a final constructionas shown in FIG.
 16. 67. The method of claim 64, wherein said transgeneis tyrosine hydroxylase.
 68. The method of claim 64, wherein said donorcells are cells from the same species of mammal receiving the cells. 69.The method of claim 1, wherein said donor cells are cells from adifferent species of mammal receiving the cells.
 70. The method of claim69, wherein immunological reactions to said donor cells are suppressed.71. The method of claim 70, wherein an immunosuppression agent isadministered.
 72. The method of claim 1, wherein said transgene is agene coding for tyrosine hydroxylase.
 73. The method of claim 1, whereinsaid transgene is a gene coding for NGF.
 74. The method of claim 1,wherein said transgene is a gene coding for ChAT.